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Expression and regulation of B7 on antigen presenting cells

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Expression and regulation of B7 on antigen presenting cells

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, som e thesis and dissertation copies are in typewriter face, while others may be from any type of com puter printer. The quality of this reproduction is dependent upon the quality o f the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete m anuscript and there are missing pages, th ese will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., m aps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. ProQ uest Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Expression and Regulation o f B7 on Antigen Presenting Cells By Zheng Liu A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment o f the Requirements for the Degree DOCTOR OF PHILOSOPHY (PHARMACEUTICAL SCIENCES) M ay 2000 Copyright 2000 Zheng Liu Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 3018019 ___ ® UMI UMI Microform 3018019 Copyright 2001 by Bell & Howell Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CALIFORNIA 90007 This dissertation, written by Zheng L iu under the direction of /tex. Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of re quirements for the degree of DOCTOR OF PHILOSOPHY Dean o f Graduate Studies Date .... f^ P .F .f.-L 29.9.9. DISSERTATION COMMITTEE ............ f ....... 00 V Chairperson Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Zheng Liu Committee Chair: Hermann von Grafenstein Expression and Regulation of B7 on Antigen Presenting Cells Antigen-specific T cell activation requires the engagement o f T cell receptor (TCR) with antigen-derived peptides bound to MHC molecules as well as the engagement of costimulatory molecules. The most extensively characterized pathway o f costimulation is the interaction o f CD28 on T cells with B7 on antigen presenting cells. In this study, the expression and regulation o f B7 on macrophages and their costimulatory function in T cell proliferation were studied. The results show that B7 expression on macrophage subpopulations is regulated differentially in response to LPS stimulation. Alveolar macrophages did not respond to LPS and a combination o f various stimuli, whereas peritoneal macrophages responded to LPS in a dose-dependent manner in B7-2 expression. However, alveolar macrophages released more T N F-a than peritoneal macrophages in response to LPS. The influence o f T cells on the expression of B7 on thioglycollate elicited peritoneal macrophages was further investigated. In contrast to B cells, stimulating macrophages wdth immobilized anti-CD40 did not up-regulate B7 expression. On the other hand, LFN-y had an effect similar to that of LPS in inducing B7 expression on macrophages. B7 molecules on each macrophage were enumerated using microbeads for calibration o f flow cytometric dada. About 20,000 B7-2 molecules on each macrophage were sufficient to costimulate T cells for their TCR dependent proliferation in conjunction with appropriate concentrations o f anti-CD3 antibody and ratios o f macrophages to T cells. T cell proliferation was almost completely inhibited by the addition of anti-B7-2 antibody and Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. moderately inhibited by anti-B7-l antibody. As an alternative m ethod to flow cytometry to measure B7 expression on Fc receptor bearing cells, a cell-ELISA method was developed by forming a conjugate between the F(ab ’)2 fragment o f an anti-B7-2 antibody and p- galactosidase using a water soluble heterobifunctional crosslinker. The measurement o f B7-2 on B cells was comparable with both methods. These results indicate that macrophage subpopulations m ay have distinct functions and the regulation o f B7 expression on such cells is more dependent on microbial substitutes than T cells, and B7-1 and B7-2 may have different function for T cell activation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION For my family Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACK NO W LEDG M ENTS I would like to express my deep gratitude to my advisor, Dr. Hermann von Grafenstein, for his guidance, enthusiasm and encouragement during the entire course o f my graduate studies. Also I would like to thank my committee members, Dr. Minnie McMillan and Dr. Ian Haworth, fo r their valuable time in providing advice and helpful comments fo r the completion o f this dissertation. I really appreciate my colleagues fo r their suggestions, help and pleasant cooperation. Finally, my warmest gratitude goes to my family members who gave me consistent spiritual support that made it possible fo r me to have today’ s accomplishment. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS DEDICATION..................................................................................................................... ii ACKNOW LEDGM ENTS.................................................................................................iii LIST OF FIGURES.............................................................................................................vii LIST OF T A B L E S..............................................................................................................ix LIST OF SCHEM ES.......................................................................................................... x LIST OF ABBREVIATIONS.......................................................................................... xi CHAPTER ONE INTRODUCTION...........................................................................1 1.1 Two signal model for T cell activation..................................................... 2 1.2 Antigen presenting cells (A PCs) ..................................................... 6 1.3 B7-1 and B7-2.................................................................................................7 1.4 CD28 and CTLA -4........................................................................................ 10 1.5 Signal transduction in T cell activation.....................................................14 1.5.1. Signaling pathways through T cell receptor (T C R )............... 14 1.5.2. Signaling pathways through C D 28............................................17 1.5.3. Signaling pathways through CTLA-4.......................................20 1.6 Accessory molecules other than C D 28..................................................... 21 1.7 Significance o f studying costimulatory m olecules................................. 25 1.7.1. Autoiinmunity................................................................................25 1.7.2. Transplantation..............................................................................27 1.7.3. Vaccine development and immunotherapy for cancer 28 1.8 Scope o f the dissertation...............................................................................30 CHAPTER TW O COMPARISON OF B7 EXPRESSION ON MACROPHAGE SUBPOPULATIONS....................................33 2.1 Introduction..................................................................................................... 34 2.2 Materials and M ethods................................................................................. 40 2.2.1 Animals........................................................................................... 40 2.2.2 Cell culture m edium .................................................................... 40 2.2.3 Reagents..........................................................................................40 2.2.4. Preparation o f m acrophages........................................................41 2.2.5. FACS analysis o f B7-1 and B7-2...............................................42 2.2.6. ELISA for T N F -a .........................................................................42 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.3 R esults............................................................................................................ 44 2.3.1 Comparison o f B7 expression on alveolar macrophages (AMs), resident peritoneal macrophages (rPMs) and thioglycollate elicited peritoneal macrophages (PM s)..................................................44 2.3.2 Low expression o f B7 on alveolar macrophages....................44 2.3.3 TN F-a production by macrophage subpopulations............... 45 2.4. Discussion..................................................................................................... 50 CHAPTER THREE REGULATION OF B7 BY SIGNALS FROM T CELLS........................................................................................ 53 3.1 Introduction................................................................................................... 54 3.2 Materials and Methods................................................................................60 3.2.1 Animals, cell culture medium, macrophage preparation, FACS analysis of B7-1 and B7-2....................... 60 3.2.2 Preparation o f B cells...................................................................60 3.2.3 Measurement o f CD 40.................................................................61 3.3 R esults........................................................................................................... 61 3.3.1 Effect of CD40 crosslinking on B7 expression..................... 61 3.3.2 Soluble effectors on B7 expression in macrophages............62 3.4 Discussion..................................................................................................... 70 CHAPTER FOUR FUNCTIONAL COSTIMULATORY ACTIVITY OF M ACROPHAGES.......................................... 74 4.1 Introduction................................................................................................... 75 4.2 Material and m ethods.................................................................................78 4.2.1 T cell preparation........................................................................ 78 4.2.2 Immunofluorescenct staining of microbeads..........................78 4.2.3 Construction o f standard calibration curve.............................79 4.2.4 Determination o f binding capacity o f anti-B7 antibodies to m acrophages.........................................................B O 4.2.5 Proliferation assay o f T cells.................................................... 80 4.3 R esults........................................................................................................... 81 4.3.1 Quanitfication of B7 expression on resident and thioglycollate elicited peritoneal macrophages...................... 81 4.3.2 Costimulation by macrophages of anti-CD3 induced T cell proliferation...................................................... 82 4.3.3 Inhibition of T cell proliferation by anti-B7 antibodies 95 4.4 Discussion..................................................................................................... 96 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER FIVE DEVELOPMENT OF A CELL ELISA METHOD TO MEASURE B7 EXPRESSION.........................102 5.1 Introduction......................................................................................................103 5.2 Materials and m ethods.................................................................................. 108 5.2.1 Reagents and antibodies.............................................................. 108 5.2.2 Purification o f antibodies............................................................ 108 5.2.3 Preparation o f F(ab’)2 fragments................................................109 5.2.4 Synthesis o f the antibody-P-galactosidase conjugate.............110 5.2.5 Assay o f P-galactosidase-antibody conjugate formation by capture ELISA .......................................................111 5.2.6 Direct (one-step) ELISA to measure B7 on B cells................111 5.3 R esults.......................................... 112 5.3.1 Formation o f antibody-P-galactosidase conjugates using a heterobifunctional crosslinker.................. 1 1 2 5.3.2 M easurement o f B7-2 expression on B cells using anti-B7-2 mAb F(ab’)2-P-galactosidase conjugate and comparison with that using flow cytometry......................113 5.3.3 Specificity o f the anti-B7-2 mAb F(ab’)2-P~ galactosidase conjugate................................................................ 119 5.4 Discussion........................................................................................................ 119 CHAPTER SIX SUMMARY AND PERSPECTIVES........................................... 124 REFERENCES.................................................................................................................... 129 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES Figure 2.1 Comparison o f B7 expression on different macrophage subpopulations..............................................................................................46 Figure 2.2 Expression o f B7 on alveolar macrophages............................................. 47 Figure 2.3 Time course o f B7 expression on thioglycollate elicited peritoneal macrophages (P M ) ...................................................... 48 Figure 2.4 TN F-a production by macrophage subpopulations............................... 49 Figure 3.1 Different effect of crosslinking CD40 on B7 expression in B cells and thioglycollate elicited peritoneal m acrophages.....................6 6 Figure 3.2 Time course o f B7-2 expression in B cells and thioglycollate elicited peritoneal macrophages after crosslinking o f C D 40..............65 Figure 3.3 Increased CD40 expression on macrophages did not enhance the B7-2 expression...................................................................... 6 6 Figure 3.4 The effect o f IFN-y on B7-2 expression in thioglycollate elicited peritoneal macrophages................................................................ 67 Figure 3.5 The effect o f GM-CSF on B7 expression in thioglycollate elicited peritoneal m acrophages...................................... 6 8 Figure 3.6 The effect of PGEt on LPS induced B7 expression in thioglycollate elicited peritoneal macrophages...................................... 69 Figure 4.1 Histogram o f microbeads staining............................................................. 84 Figure 4.2 Standard calibration curve derived from Figure 4 .1 .............................. 85 Figure 4.3 Expression o f B7 on resident peritoneal m acrophages..........................8 6 Figure 4.4 Histogram o f microbeads staining............................................................. 87 Figure 4.5 Standard calibration curve derived from Figure 4 .5 .............................. 8 8 Figure 4.6 Expression o f B7 on thioglycollate elicited peritoneal macrophages.................................................................................................. 89 vii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 4.7 Dependency o f T cell proliferation on num ber o f macrophages and concentrations o f anti-C D 3.......................................90 Figure 4.8 T cell proliferation correlates with B7 level on macrophages.............91 Figure 4.9 Inhibition o f T cell proliferation by anti-B7 antibodies........................ 92 Figure 4.10 Measurement o f the average number o f B7 molecules on each macrophage..........................................................................................93 Figure 5.1 Capture ELISA performed to assay formation o f GL1 F(ab’)2-P-galactosidase conjugate...................................................115 Figure 5.2 Measurement o f B7-2 expression on ex vivo B cells, B cells cultured in medium and B blasts by cell-ELISA using a direct m ethod.................................................................................. 116 Figure 5.3 Comparison o f cell-ELISA (a) and flow cytometry (b) to measure B7-2 expression on ex vivo B cells, B cells cultured in medium and B blasts............................................................... 117 Figure 5.4 Competition assay to test the specificity o f GL1 F(ab’)2-[3-galactosidase conjugate...................................................118 viii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1.1 Properties o f antigen presenting c ells...................................................... 4 Table 1.2 Some pattem-recognition receptors (PRRs) on A P C s..........................5 Table 4.1 Measurement o f B7 on thioglycollate elicited peritoneal macrophages................................................................................................. 94 ix Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF SCHEMES Scheme 1.1 Signal transduction in T cell activation................................................. 13 Scheme 2.1 Structure o f LPS o f Salmonella enterica...............................................36 Scheme 3.1 The role o f CD40-CD40L in T cell activation..................................... 59 Scheme 5.1 Structure o f sulfo-SMCC and the reactions to form antibody-enzyme conjugate........................................................................ 114 x Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF ABBREVIATIONS ABC: antibody binding capacity AM: alveolar macrophages AP: alkaline phosphatase APCs: antigen presenting cells A-SMase: acidic sphyngomyelindase CPRG: chlorophenolred-P-D-galactopyranoside CTL: cytotoxic T lymphocytes CTLA-4: cytotoxic lymphocyte-associated molecule-4 DAG: diacylglycerol EAE: experimental autoimmune encephalomyelitis ELISA: enzyme-linked immunosorbent assay ERK: extracellular-signal-regulated kinase FBS: fetal bovine serum FcR: Fc receptor FITC: fluorescein isothiocyanate GM-CSF: granulocyte-macrophage colony-stimulating factor GVHD: graft-versus-host disease HRP: horseradish perodixase ICAM: intercellualr adhesion molecule EP 3: inositol 1,4,5-trisphosphate ITAM: immunoreceptor tyrosine-based activation motifs JNK: Jun N-term inal kinase LBP: LPS binding protein LFA-1: lymphocyte function-associated antigen 1 LPS: lipopolysaccharide mAb: monoclonal antibody MAP: mitogen-activated protein MBS: m-maleimidobenzoyl-iV-hydroxysuccinimide ester MHC: m ajor histocompatibility complex NF-kB: nuclear factor kB NF-AT: nuclear factor o f activated T cells NOD: non-obese diabetic OPD: O-phenylenediamine PAMPs: pathogen-associated molecular patterns PE: phycoerythrin PECs: peritoneal exudate cells PGE2: prostaglandin E2 PEP2: phosphatidylinositol 4,5 bisphosphate PKC: protein kinase C PLCyl: phospholipase Cyl xi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. PM: thioglycollate elicited peritoneal macrophages PRRs: pattem-recognition receptors PTK: protein tyrosine kinase rPM: resident peritoneal macrophages SH2: Src homology region 2 SLAM: signaling lymphocyte activation molecule Sulfo-SMCC: sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-l- carboxylate TCM: tissue culture medium TCR: T cell receptor TNFR: tumor necrosis factor-a receptor TRAF: tumor necrosis factor-a receptor associated factors Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER ONE INTRODUCTION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1. Two-signal model for T cell activation Adaptive immune responses involve the activation o f naive lymphocytes that recognize foreign antigens with their antigen receptor molecules. Rare antigen-specific naive lymphocytes that recognize antigen have to proliferate to increase their frequency before they differentiate into armed effector cells that are able to mediate the elimination o f the pathogen that provokes the response. However, proliferation followed by differentiation into effector cells is not the only possible result of antigen recognition by a lymphocyte receptor: naive lymphocytes can be activated, inactivated, or triggered to die through their receptors. The fate of T cells is determined by the signals they received from antigen presenting cells (APCs). Bretscher and Cohn (Bretscher and Cohn. 1970) first proposed a two-signal model of B lymphocyte activation that was later modified by Lafferty and Cunningham (Lafferty and Cunningham. 1975) for T cell activation. The essential features o f these models are that activation o f lymphocytes requires an antigen-specific "signal one” as well as a second antigen non-specific event termed “signal two" (Janeway and Bottomly. 1994: Linsley and Ledbetter. 1993; Schwartz, 1992). For T cells, “signal one" is defined as the occupancy of the T-cell receptor (TCR) by a complex formed between an antigenic peptide and a major histocompatibilility complex (MHC) molecule on the APC surface. “Signal two” is delivered by soluble costimulatory factors or by a ligand molecule i Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. on the APC surface, which binds to a distinct receptor on the T cell surface. Signal two or costimulation is defined by the ability to deliver an independent signal that, in association with the TCR signal, modulates proliferation, cytokine production and prevention o f anergy of T cells. According to the two-signal model, T cells triggered through the TCR (signal one) in the absence o f costimulation (signal two) become anergic, i.e. they cannot respond to their specific antigen under optimal conditions o f stimulation. Signal two alone does not induce a T cell response. T cell activation and clonal expansion occur when both two signals are received simultaneously. A number o f agents can specifically or nonspecifically induce T cell activation, resulting in cytokine production, cytokine receptor expression, and ultimately proliferation o f the activated T cells. One o f the nonspecific means to activate T cells is to crosslink the TCR complex component CD3. the cluster o f five chains transducing signal to the interior o f the cell when antigen-binding has occurred, by anti-CD3 antibody. This will provide signal one to T cells. The addition of APCs provide signal two for T cell proliferation as well as crosslink (via their Fc receptors) monoclonal anti-CD3 antibody bound to CD3. This anti- CD3 induced T cell proliferation has been a commonly used assay for T cell function. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1.1. Properties of antigen presenting cells Cell type Antigen uptake MHC II Co- stimulator Critical antigens 1 1 F+ +-H- -to +++ Toxin, viruses. 1 j v d l l J Specific Ig Constitutive bacteria receptor Macrophages +-H- - to +++ - to +++ Intracellular Phagocytosis pathogens Dendritic - to i i-1-1 ■ !■ + + + - to ++++ Vi mses cells Depending on Constitutive Depending on maturation maturation stage stage Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 1.2. Some pattem-recognition receptors (PRRs) on APCs Receptor Location Ligands Function Mannose receptor Macrophages Terminal mannose Phagocytosis DEC 205 Dendritic cells Terminal mannose Phagocytosis CD 14 Macrophages LPS Clearance of microbes, induction o f proinflammatory cytokines RP 105 B cells unknown Mitogenic for B cells CD35 (CR1) Macrophages C3b. C4b Enhances C3b and C4b cleavage CD21 (CR2) B cells, dendritic cells iC3b. C3d Augments B cell activity C D llc , CD 18 (CR3) Macrophages, dendritic cells iC3b. LPS Adhesion. phagocytosis Scavenger receptors Macrophages Bacterial cell walls Phagocytosis, bacterial clearance Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2. Antigen presenting cells (APCs) During an immune response. APCs are the first cells to encounter and recognize antigens. There are three types o f professional antigen presenting cells (APCs), B cells, macrophages, and dendritic cells (Janewav, 1992). APCs are not only' able to present specific antigenic peptides in the context o f MHC complex to T cells, but also provide costimulation to T cells for their full activation. The properties of these APCs are listed in Table 1.1. It has been suggested by Charles Janeway that the APCs should be able to distinguish self from non-self, and provide the costimulation only when pathogens are present. This is achieved by a set o f non-clonal receptors involved in innate immune responses (Janeway. 1992). The immune system has evolved under selective pressure imposed by pathogens. As a result, all multicellular organisms have developed the ability to recognize invading microbes and to eliminate them efficiently without causing damage to self. APCs express so called pattem-recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) rather than particular structures. According to this hypothesis PRRs are perfectly able to discriminate self from non-self pathogen-associated staictures not produced by the host because they evolved to recognize non-self. Therefore, the signals induced by the ligation o f PRRs would signal the presence o f a pathogen and would be interpreted as such by the rest o f the immune system. In other words, the recognition of pathogens by PRRs induces the expression o f costimulatory 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. molecules on the surface o f APCs and facilitates pathogen uptake and degradation. The latter function was then further adapted to present unique signals from pathogens in the form o f MHC-bound peptides to the receptor on T cells. Thus, the presence o f the pathogen is recognized by the APCs by the binding o f one or more of the PRRs to PAMPs. and the specific pathogen-derived antigens are presented by the same APC through the processing and presentation o f peptides specific to that pathogen. This whole mechanism ensures that a T cell will normally receive both signals necessary for activation only if the peptide recognized by the TCR is derived from the pathogen that initially induced the costimulatory activity (Fearon and Locksley. 1996: Medzhitov and Janewav. 1997). Some PRRs on APCs are listed in Table 1.2. 1.3. B7-1 and B7-2 Among the costimulatory molecules, the B7 family appears to be unique, since ligation by B7 on APCs o f their counter receptor CD28 on T cells is necessary and sufficient to prevent the induction o f anergy (Boussiotis et al.. 1994; Lenschow et al., 1996). Two members of the B7 family. B7-1 (CD80) and B7-2 (CD 8 6 ) have been cloned and functionally characterized. They both are members o f the immunoglobulin gene superfamily with an Ig-V-like and an Ig- C2-Iike domain. Comparison o f the amino acid sequences of B7-1 and B7-2 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reveals 25% amino acid identity. The cytoplasmic domains o f B7-1 and B7-2 are markedly different witii the cytoplasmic domain of B7-2 being longer and containing potential phosphorylation sites (Azuma et al.. 1993: Freeman et ah. 1989; Freeman et al., 1993). Under physiologic conditions, B7 expression is tightly controlled and is restricted to professional APCs. including dendritic cells, macrophages and activated B cells. Resting B cells express no detectable B7-1 and very low levels o f B7-2. Both B7-1 and B7-2 are upregulated following B cell activation with agents such as LPS, concanavalin A or cAMP. However, dramatic differences exist in the kinetics and the signals that control B7-1 and B7-2 expression. The induction of B7-2 occurs within 6 h o f stimulation, with maximal levels of expression achieved between 18 and 24 h. In contrast, B7-1 expression is not detected until 24 h post stimulation and does not reach maximal levels until 48 to 72 h later. Furthermore, activated B cells and dendritic cells express quantitatively higher levels of B7-2 than B7-1 (Hathcock et ah. 1994: Lenschow et ah, 1993). The regulation of B7-1 and B7-2 expression on B cells is controlled by cell-cell interactions and cytokines. The cross-linking of surface Ig byr anti-Ig or antigen rapidly induces B7 on B cell surface (Lenschow et ah. 1994). A number of cytokines have been shown to differentially regulate B7-1 and B7-2 expression. IL-4 is one of the most potent inducers o f B7-2 and. to a lesser extent, B7-1 on B cells (Stack et ah, 1994). INF-y increases the expression o f B7- 2 on B cells and peritoneal macrophages. IL-10 blocks both B7-1 and B7-2 up- 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulation on peritoneal macrophages and dendritic cells (Buelens et al.. 1995: Willems et al., 1994). Whether B7-1 and B7-2 have identical, overlapping or distinct functions is not completely understood. However, the distinctive properties o f these two molecules, including the different levels and kinetics of expression on various APCs as well as the different avidity to their ligands, indicate that B7-1 and B7-2 may differentially control the immune response. While B7-2 dominates in primary responses. B7-1 may be important in the control o f an ongoing response (Harris and Ronchese, 1999). Numerous in vitro and in vivo studies have shown that B7-1 and B7-2 have an important role in Thl and Th2 differentiation. T n a system using B7-1 and B7-2 transfected CHO cells. Freeman et al. showed that B7-1 signals preferentially promote development of Thl cells, while B7-2 signals promote Th2 cell development (Freeman et al.. 1995). This result was confirmed by an in vivo study using a model o f experimental autoimmune encephalomyelitis (EAE) (Kuchroo et al., 1995). In this system, treatment with anti-B7-l antibody reduces the incidence o f disease by promoting Th2 cell development. Conversely, treatment with anti-B7-2 antibody increases disease severity by promoting the development of Thl cells. However, some other studies have yielded contradictory results. A study investigating the roles of B7-1 and B7-2 using an autoimmune model o f diabetes, mediated by Thl cells, shows that blockade o f B7-1 costimulation increases disease incidence, presumably by increasing development of Thl cells. Blockade of B7-2 prevents the onset o f 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. diabetes by down-regulating Thl cell responses. This study indicates that B7-1 and B7-2 promote Th2 and Thl cell responses, respectively, and is in direct contrast to the data obtained in the EAE model (Lenschow et al., 1995). Regardless o f whether B7-1 and B7-2 costimulation directly modulates Thl/Th2 differentiation, the above studies establish that B7-1 and B7-2 can have different effects on T cell immune responses. 1.4. CD28 and CTLA-4 CD28 and cytotoxic T lymphocyte-associated molecule-4 (CTLA-4. CD 152) are the two counter-receptors of B7-1 and B7-2. CD28 and CTLA-4 are glycoproteins that belong to the immunoglobulin supergene family and are expressed as homodimers on T cells (Lenschow et al.. 1996). Both molecules bind to ligands B7-1 and B7-2 via the MYPPPY m otif in the immunoglobulin domain. CTLA-4. however, has a 10-fold higher affinity and a 100-fold higher avidity for B7 ligands compared to CD28 and exhibits distinct binding kinetics (Metzler et al., 1997; van der Merwe et al.. 1997). The cytoplasmic tails of CD28 and CTLA-4 possess tyrosine-containing motifs postulated to be involved in signal transduction and protein trafficking. These two molecules have distinct expression patterns on T lymphocytes. CD28 is constitutively expressed at relatively constant levels on the cell surface of CD4 and CD8 T cells, with small fluctuations in ceil surface expression following T cell activation (Lenschow et 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al., 1996). In contrast, the CTLA-4 protein is not detectable on resting T cells and it can be detected on the cell surface 24 h after T cell activation in both vitro and in vivo. The CTLA-4 molecule reaches peak expression 36-48 h after activation (Krummel and Allison, 1995: Walunas et al., 1994). The majority o f the CTLA-4 protein is localized inside the cell, particularly in the peri-nuclear Golgi, even at the time o f maximal CTLA-4 expression on the cell surface o f activated T cells. Intracellular CTLA-4 traffics to the plasma membrane at the site of TCR-APC interaction and is quickly endocytosed (Leung et al., 1995; Linsley et al., 1996). The cytoplasmic tail of CTLA-4 contains a tyrosine-based YXXtj) (in the single letter code for amino acids where X is any amino acid and ( j ) is any bulky hydrophobic residue) internalization signal consensus sequence that mediates interaction with the adapter proteins AP-I. AP- 2 and AP-3 involved in protein sorting. In addition to the differences o f expression and the affinity to B7. CD28 and CTLA-4 display opposite functions in the T cell response. CD28 is the primary positive T cell costimulatory molecule, as defined by the ability to enhance T cell activation and IL-2 production in the presence o f TCR stimulation. CD28 engagement enhances T cell survival and can prevent anergy induction (Lenschow et al., 1996). CD28 mediated stimulation enhances the expression o f other T cell surface molecules like CD40L, which has been shown to be vital for the full activation o f naive CD8 T cells and T cell effector functions as well 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Yang and Wilson, 1996). These findings underline the importance o f positive costimulation in facilitating effective T cell responses. Studies during the past several years using blocking antibody and CTLA-4 knock-out mice indicated the negative costimulatory role o f CTLA-4 in regulating T cell activation. Blockade o f CTLA-4 with anti-CTLA-4 antibody augments T cell responses in vitro and in vivo (Chambers and Allison. 1997). Mice deficient in CTLA-4 display polyclonal T cell expansion that results in a fatal lymphoproliferative disorder and the mice die at 3-4 weeks of age. Most o f the peripheral T cells in these mice have an activated phenotype, and there is a 4-fold increase in the proportion o f T cells undergoing cell division in these animals (W aterhouse etal., 1995). It is clear that the balance of positive and negative costimulation plays a critical role in the production o f effective immune responses and for maintaining T cells homeostasis. However, their regulation and the integration o f these signals with TCR-mediated signals are just beginning to be elucidated. Understanding the balancing act o f T cell activation will be necessary for effective manipulation of clinically relevant effector T cell responses and the generation of long-term antigen-specific T cell memory. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TCR/ITAMs CD28 Lck Lck/fyn ZAP-70 A-SMase Ras PLCyl Ceramide PKC Raf PIP2 MEK IP3 + DAG Ras/Raf MEK MAPK JNK/ERKI/2 PKC Calcineurin NF-ATc Jun/Fos API NF-kB Cytokine genes (e.g. IL-2) Nucleus Scheme 1.1. Signal transduction in T cell activation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.5. Signal transduction in T cell activation The signal transduction in T cells is complex. This is necessary because signal integration by T cells must ensure responsiveness to antigenic challenge and tolerance. Depending on which pathway is activated. T cells may differentiate into effector cells, the effector cells may be activated and proliferate, or alternatively these cells may become anergy or die by apoptosis. Many studies have been done aiming at identifying the effector pathways that possibly are triggered during T cell activation. 1.5.1. Signaling pathways through T cell receptor (TCR) The TCR is associated with the CD3 cluster, which is composed o f the invariant subunits y. 5 and s, as well as with the C:C homodimer chains. These chains, representing the signal transducing complex associated with the TCR. do not possess intrinsic enzymatic activity. However, they contain immunoreceptor tyrosine-based activation motifs (ITAM) in their cytoplasmic tail (Baniyash et al.. 1988; Osman et al., 1995; Osman et al.. 1996). The ITAMs can be tyrosine phosphorylated, leading to the recruitment of protein tyrosine kinase (PTK). These PTKs trigger phosphorylation cascades that transduce the activation signals from the membrane to the nucleus as represented in Scheme 1.1. IL-2 gene transcription, which is a key event in T cell activation and proliferation, is regulated by the coordinated action o f multiple factors including nuclear factor o f 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activated T cells (NF-AT), activated protein-l (A P-l), nuclear factor kB (NF-kB). and octamer protein-1 (Oct-1) (Jain et al., 1995). There are at least two purine- rich binding sites for NF-AT family members in the mouse IL-2 promoter. The distal site on the IL-2 promoter is bound with high affinity by a complex formed by NF-AT proteins and an AP-l-type dimer composed o f Jun and Fos proteins. The c-Jun homodimers or the c-Jun/c-Fos heterodimers stabilize the binding o f NF-AT to DNA (Jain et al., 1995). Rapid phosphorylation of the ITAM by the Src family PTK p56/c A . and potentially by p59^". permits TCR association with the Syk family kinase ZAP-70 via two Src homology-2 (SH2) domains, leading to the phosphorylation o f this kinase and its activation (Chan et al.. 1995; Chan et al.. 1992: van Oers et al.. 1996). Phosphorylation o f the TCR complex allows the association of several ‘^adaptor” molecules, leading to several signaling cascades. One o f these cascades involves phosphorylation and activation o f phospholipase Cyl (PLCyl) (Weber et al., 1992; Weiss et al., 1991), which cleaves phosphatidylinositol 4.5 bisphosphate (PIP2) to generate diacylglycerol (DAG) and inositol l.4.5-trisphosphate (IP3). which, respectively, activate PKC and trigger an increase in the concentration o f intracellular calcium. One o f the down-stream effects o f the rapid and sustained increase in calcium concentration is the activation o f the calmodulin/calcium- dependent serine/threonine phosphatase calcineurin. This phosphatase dephosphorylates the transcription factor NF-AT and the dephosphorylated NF- 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AT translocates from cytoplasm to the nucleus where it participates in IL-2 gene transcription (Crabtree and Clipstone. 1994). The second signaling pathway necessary for NF-AT activation involves the low molecular weight G protein p 2 lra\ which, in quiescent T cells, exists as its "inactive’" GDP-bound form and which, upon TCR engagement, rapidly and stably accumulates as its "active” GTP-bound complex (Izquierdo Pastor et al.. 1995). Regulation o f NF-AT by p21ra s can be explained through its effects on AP- 1. Activation o f AP-l (Foletta et al., 1998). which binds the IL-2 promoter both directly and as a component o f an NF-AT complex, results from increased expression and phosphorylation o f Fos and Jun family proteins. It has been established that regulation of c-fos gene transcription is mediated by the nuclear factor Elk-1, which is phosphorylated and activated by ras-dependent signal cascades involving the extracellular-signal-regulated kinase (ERK) and Jun N- terminal kinase (JNK) (Marais et al.. 1993). On the other hand, c-jun is also regulated by JNK for which it appears to be a direct substrate. ERK 1/2. JNK. and another pathway involving the kinase p38. which represent parallel kinase cascades initiated at the ras level, are commonly termed mitogen-activated protein (MAP) kinase pathway (Su and Karin, 1996). In human T cells, at least two MAP kinases. Erk-1 and Erk-2, are activated through the ras pathway in response to occupancy of the TCR (Izquierdo et al.. 1993). Activation o f ERK1/2 is induced by phosphorylation mediated by MAP 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. kinase kinase 1/2 (M EK-1/2). the activity o f which is itself regulated through phosphorylation by a MAP kinase kinase kinase. The serine/threonine protein kinase Raf-1, which couples p 2 lras and interacts with MEK-1 to form a ternary signaling complex, appears to be this MAP kinase kinase kinase (Howe et al.. 1992). It has been shown that constitutively active Raf-1 can act in stimulating ERK-2 and in synergy with calcium signals, in inducing IL-2 gene expression. The p21ra s/MEK/ERK pathway, shown in neuronal cells and fibroblasts, has been hypothesized to be similarly involved in NF-AT activation and IL-2 gene transcription in T cells (Marais et al., 1993). 1.5.2. Signaling pathways through CD28 A key feature o f the costimulatory signal provided by CD28 is that in conjunction with a TCR stimulus, it allows high-level production of IL-2 as a result of increased levels o f transcription and mRNA stabilization, and provides an essential survival signal for T cells, thereby preventing apoptosis or the induction of anergy that may occur in response to signal one alone (Lenschow et al., 1996; Schwartz, 1996). The ligation of CD28 has been shown to activate PLCyl and calcium mobilization (Ledbetter and Linsley. 1992). This receptor does not have any intrinsic enzymatic activity. However, it can be phosphorylated on tyrosine residues on its cytoplasmic tail, allowing the binding o f different signal transducing molecules such as PI 3-kinase, growth factor- bound protein-2 (Grb-2) from the Grb/son of sevenless (Grb-2-SOS) complex, 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and IL-2 inducible T cell kinase (Itk). The Src family kinases p56lc k and p59^7 '. have also been shown to physically associate with the receptor, and p56/c*. demonstrates a remarkable phosphorylating specificity for the pTyrI7j-M et-Asn- Met motif within the CD28 cytoplasmic tail. This phosphorylated motif on T yr1 /J binds to the SH2 domain of the 85 kDa regulatory subunit o f PI 3-kinase (Pages et al., 1994). The recruitment and activation o f PI 3-kinase is essential in the CD28- induced response (Rudd. 1996: Ward et al.. 1996). PI 3-kinase is a heterodimer composed o f an adaptor subunit (p85) linked to a catalytic domain (pi 10) that mediates the formation o f D-3 phosphoinositide lipids by transferring the terminal phosphate o f ATP to the D-3 position o f the inositol ring o f phosphatidvlinositol. phosphatidylinositoi 4-phosphate, and phosphatidylinositol 4.5-bisphosphate. generating PI 3-P. PI 3 ,4 -P2, and PI 3 ,4 ,5 -P3, respectively. PI 3.4-Pi and PI 3.4.5- P3 can activate the 8 , s, r|. and C , isoforms o f the serine/threonine kinase, protein kinase C (PKC), as well as the kinase encoded by the proto-oncogene Akt. PI 3- kinase may also activate or may be activated by p21ra ~ s (Hu et al.. 1995: Rodriguez-Viciana et al., 1994). Grb-2-SOS. which binds to CD28. appears to be a good candidate to couple CD28 with the p21n L S pathway, however, the physiological relevance of this effect is not clear and may not be essential in CD28 costimulation. The other CD28 binding protein is Itk. also known as EMT. a Tec family kinase that is expressed in T cells (Siliciano et al.. 1992). CD28 activation o f this PTK has been shown to be dependent upon functional Lck. The targets o f such a PTK in CD28 signaling are poorly defined, but a recent study has 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. shown that EMT/Itk can phosphorvlate all four tyrosines o f the CD28 tail, in contrast to Lck, which phosphorylates only tyrosine 173 (King et al.. 1997). Another pathway triggered upon CD28 ligation includes activation o f an acidic sphyngomyelindase (A-SMase). which results in the generation o f phosphorylcholine and ceramide (Boucher et al.. 1995). Targets o f the latter compound include the Ras/Raf pathway (Yao et al.. 1995). JNK (Westvvick et al.. 1995), and PKCC (Lozano et al.. 1994). The signaling cascade that bridges these early events triggered upon CD28 engagement with the late ones, directly affecting activation o f nuclear factors regulating cytokine gene transcription is still not solved completely. However, recent studies using transfected cells and A-SMase inhibitors suggest that AP-l and NF-tcB are direct targets for CD28 signals and that both PI 3-kinase and A-SMase activation are involved in their generation (Edmead et al.. 1996). Moreover, evidence is accumulating that PI 3- kinase may have direct effects on the regulation of MAP kinase pathways including ERK and JNK (Ward et al.. 1996). Indeed, fas transcription can be activated by a constitutively active pi 10 mutant of PI 3-kinase (Hu et al.. 1995). since fos activation is Ras dependent, this suggests that PI 3-kinase can modulate the p21ra s pathway and, consequently, the ERK cascade. Moreover, Rac-1, which has been shown to regulate the kinase cascade leading to JNK activation, also appears to be a downstream effector of PI-3-kinase. It is noteworthy that activation o f JNK in T cells requires costimulation of TCR and CD28 by their respective antibodies. This result led to the conclusion that JNK activation could 19 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. be a nodal event between the TCR induced signaling pathway and the CD28 mediated costimulation pathway, resulting in IL-2 production. This is strongly supported by recent data, showing that Rac-1 cooperates with Syk tyrosine kinase to synergistically increase JNK activity as well as AP-l and NF-AT transcriptional activities (Jacinto et al., 1998). 1.5.3. Signaling pathways through CTLA-4 The signaling pathways of CTLA-4 have not been fully elucidated. CTLA- 4 has a short cytoplasmic tail that is 100% conserved between species and contains two tyrosine-containing motifs. YVKM and YFIP (Thompson and Allison, 1997). The YVKM m otif plays a pivotal role in the tightly controlled localization o f CTLA-4 as well as in the interaction with key signaling molecules. The nonphosphorylated YVKM motif can associate with l i - 2. one of the subunits of the clathrin-associated adaptor complex AP-2; this complex is responsible for continuous translocation o f CTLA-4 to and from the cell surface. Phosphorylation of CTLA-4 releases AP-2 and results in increased cell surface expression. In a phosphorylated state, CTLA-4 can interact with the p85 subunit of phosphatidylinosito 1-3-kinase at the YVKM m otif and/or form a complex with the tyrosine phosphatase SHP-2 (Chuang et al., 1999). It has been suggested that this CTLA-4-SHP-2 interaction could negatively regulate TCR signaling by dephosphorylating key signaling proteins (Marengere et al.. 1996). Several tyrosine kinases are capable o f phosphorylating CTLA-4 during T cell activation. 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Lck, Fyn and Txk/Rlk have been shown to associate with and phosphorylate CTLA-4 on both tyrosine residues (Chuang et al., 1999). In contrast, other kinases such as ZAP-70 are not capable o f phosphorylating CTLA-4. Recent studies provide evidence for CTLA-4 interaction with the CD3-C chain o f the TCR complex intracellularly, suggesting that CTLA-4 interferes with very early TCR signaling events (Lee et al., 1998). The interaction between CD3-C and CTLA-4 does not require other T cell specific proteins or the phosphorylation of CTLA-4. 1.6. Accessory molecules other than CD28 In spite o f the large body o f evidence demonstrating that CD28 provides a critical costimulatory signal in the initiation o f T cell responses, not all T cell mediated immune responses are CD28-dependent and in humans about 50% of CD8 T cells are CD28' (Lenschow et al., 1996). These findings have contributed to the idea that for some immune responses, the antigenic stimulus is mediated by signal one alone, or alternatively, other costimulatory molecules are involved in the induction o f these immune responses. Many molecules have been identified having an accessory or costimulatory function in T cell activation based on the ability o f antibodies recognizing these molecules to augment T cell proliferation and/or cytokine production when provided in conjunction with signals through the TCR. In contrast to the costimulatory function o f CD28, these alternative 2 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accessory molecules are not able to induce high level IL-2 production, promote T cell survival or prevent apoptosis although they are capable of augmenting initial T cell activation events through TCR. They appear to act at different stages of T cell activation/differentiation or promote the development o f different effector functions (Yashiro et al.. 1998). A newly identified accessory molecule in the CD28 family is inducible costimulator (ICOS) (H utloff et al.. 1999). It is expressed only on activated T cells and shares 39% similarity with CD28 but lacks the MYPPPY m otif that is used by CD28 to bind B7-1 and B7-2. The ligands o f ICOS are not identified yet. The major difference between ICOS and CD28 is their differential induction of IL-10 and IL-2. Treating T cells with anti-ICOS antibody enhances proliferation o f T ceils as well as production o f high levels o f IL-10, IFN-y. TNF-a. and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-2. The interaction between the integrin family member lymphocyte function- associated antigen 1 (LFA-1) and its ligands intercellular adhesion molecule (IC A M )-l, -2 and — 3 is well known to be important in T cell activation (Sedwick et al., 1999). The signals through LFA-l are required for optimal cytoskeietal reorganization in interactions between T cells and APCs. However. ICAM alone together with TCR engagement induced only a very low level o f IL-2 and failed to allow clonal expansion, which implies that while LFA-1-ICAM-1 clearly plays an important role in T cell activation, it is not capable o f providing signal two in the conventional definition. Similar to LFA-1, other adhesion molecules CD-2, ~ n Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. signaling lymphocyte activation molecule (SLAM, or CDvvl50) and heat-stable antigen (HAS) may enhance proliferation o f T cells, but they cannot replace CD28 in providing the costimulation to T cells for their activation, differentiation, and prevention o f apoptosis (Davis and van der Mervve, 1996; Wu et al.. 1998). Three costimulatory members of the TNF receptor (TNFR) family are 4- 1BB (CD 137), 0X 40 (CD 134) and CD27. Their respective TNF family ligands are 4-1BBL, OX40L and CD70. 4 -IBB is a costimulatory receptor expressed primarily on activated CD4 and CD8 T cells. During primary T cell activation, its surface expression reaches its peak at 40-64 h and declines by 110 h. 4-lBBL is expressed on mature dendritic cells, activated B cells and macrophages. 4 -IBB is one of the few costimulatory receptors that can stimulate high levels of IL-2 production by resting T cells in the absence o f CD28. However. 4-1 BB only induces a level o f IL-2 production comparable with that o f the CD28 signal when the signals through the TCR are strong enough (Saoulli et ah. 1998). Even after activation, normal B cells and dendritic cells express low level o f 4-lBBL. The expression pattern o f both ligand and receptor suggest that 4 -IBB may function during the later stages of a response, to sustain T cell activation after CD28 is down-regulated and perhaps plays a role under conditions of chronic immune stimulation. 0X 40 is expressed on activated T cells, with peak expression at 24-48 h after activation and is down-regulated by 96 h. OX40L is inducible on B cells and dendritic cells via CD40 ligation. In vitro, 0X40 engagement on T cells 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. augments proliferation to suboptimal anti-CD3 and OX40-Ig partially blocks the proliferative response. OX40L augments the production o f IL-4 and IL-5 by Th2 effectors and upregulates IL-2 production but has very little effect in IFN-y production by Thl effectors. It is suggested that the OX40L synergies with B7-I to activate naive T cells. Later in the response it can act independently o f B7-1 to enhance cytokine secretion and clonal expansion o f primary Thl and Th2 effector cells, but OX40L alone is not able to provide signal two for activation o f naive T cells (Gramaglia et al., 1998). CD27 is expressed as a 55 kDa homodimer on T cells and a subset of B cells. Its expression is upregulated upon cell activation. Its ligand CD70 is expressed only transiently upon activation of B and T cells and therefore CD27- CD70 interactions are not thought to play a role in initiation o f immune responses but are expected to come into play transiently after initial T cell activation (Lens et al., 1998). It would be interesting to study a variety o f infectious agents with respect to requirements for the different costimulatory molecules. The ability o f a particular infectious agent to induce the ligands on APCs may well determine the importance of a particular costimulatory pathway in resolving an infection. Another area of interest is to elucidate the signaling pathways used by the different accessory molecules. Understanding the details o f these pathways and their integration with signals from the TCR may lead to the further clarification of their respective roles in T cell activation. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.7. Significance o f studying costimulatory molecules The B7/CD28 pathway has been extensively studied in the past few years. Its important role is not ju st limited to basic immunology, the understanding of this pathway also provides insight into the pathogenesis and treatment o f human diseases including autoimmunity, tumors and transplant rejection. The theory of costimulation has been applied as the foundation for vaccine development. 1.7.1. Autoimmunity Because costimulatory signals appear to be pivotal in determining whether recognition o f antigen by T cells leads to T cell activation or to anergy. a role for costimulation in the development of autoimmune responses has been hypothesized. The absence o f costimulators on resting tissue APCs could serve to induce and maintain T cell tolerance to self-antigen and aberrant expression of costimulators on APCs could activate self-reactive T cells, resulting in autoimmunity. Abnormal B7 expression has been found in several human autoimmune diseases. Early multiple sclerosis (MS) plaques in the CNS are associated with increased expression o f B7-1 and IL-2, but not B7-2 (Windhagen et al.. 1995). Elevated expression o f B7-1 also has been observed in monocytes. T cells and B cells within the synovial fluid or the synovia o f rheumatoid arthritis patients (Ranheim and Kipps, 1994). In patients with autoimmune thyroiditis, increased 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. numbers o f B cells expressing B7-1 were found in preparations o f intrathyroid lymphocytes (Tandon et al.. 1994). In allergic contact dermatitis and lichen plaques, B7-1 expression has been found on Langerhans cells, dermal dendritic cells and macrophages, whereas no B7-1 expression was observed anywhere in normal skin (Simon et al., 1994). Most o f the studies carried out only examined B7-1 expression, further studies are needed to assess the expression of B7-2 in human autoimmune diseases. It has been postulated that blockade of CD28-B7 interactions preferentially affects T cells that are in the process of antigen recognition. In vitro, this blockade can induce anergy and apoptosis in antigen-reactive T cells. In the setting of autoimmune diseases, such blockade might provide a means to selectively inactivate auto-reactive T cells without damaging the entire T cell repertoire. Blockade o f B7 by CTLA-4-Ig and monoclonal antibodies has been investigated in treatment o f autoimmune diseases. Injection o f CTLA-4-Ig into NIB/NZW FI mice, which develop an autoimmune disease that closely resembles systemic lupus erythematosus (SLE) in humans, prevents the production of autoantibodies (Finck et al., 1994). In non-obese diabetic (NOD) mice, administration o f CTLA-4-Ig early in the course of insulitis prevents progression to diabetes (Lenschow et al.. 1995). In experimental allergic encephalomyelitis (EAE), initiation of CTLA-4-Ig treatment the day before immunization of susceptible mice with MBP and continued for 3 weeks, prevents development o f the clinical and histological manifestations o f EAE (Klioury et al.. 1995). The 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. effect o f blocking B7 by monoclonal antibodies is complicated by the differential expression and function o f B7-1 and B7-2. The complexity may also be caused by the immunogenicity o f the antibody itself. 1.7.2. Transplantation One obstacle in organ transplantation is graft rejection. Despite great improvements in immunosuppressive therapies to prevent acute rejection, chronic graft rejection remains a significant problem. Moreover, immunosuppressive therapy is costly, and is associated with a significantly increased risk for infection, cancer and other morbidity. The progress in immunology provides a new strategy to prevent rejection: to block B7-mediated costimulation to induce tolerance. Tolerance induction may lead to prolongation of graft survival in solid organs and amelioration or prevention of graft-versus-host disease (GVHD) in allogeneic bone marrow transplantation. Several in vivo murine transplantation studies have demonstrated that blockade of the B7/CD28 pathway can be used to induce tolerance. Treatment with human CTLA-4-Ig resulted in long-lasting tolerance to human xenoantigens in mice (Lenschow et al., 1992) and suppressed antibody responses to sheep red blood cells in vivo (Linsley et al., 1992). In a murine model of bone marrow transplantation, CTLA-4-Ig treatment reduced the incidence of lethal GVHD, without effect on hematopoetic reconstitution (Blazar et al., 1994). Clinical trials 27 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. using such strategies to prevent solid organ transplant rejection and GVHD are ongoing. 1.7.3. Vaccine development and immunotherapy for cancer Tumor-specific cytotoxic T lymphocytes (CTLs) are effectors that eliminate tumors. Although tumor cells may present their antigen to CTLs. most o f the tumor cells do not express costimulatory molecules, therefore inducing anergy in the T cells specific to tumor antigens, allowing the tumor to escape its eradication by the immune system. Therapeutic strategies using augmentation of costimulation have taken four general approaches, all o f which have been evaluated in phase I. II. or III clinical trials. The first strategy is the ex vivo expansion, activation, and adoptive transfer of T cells. By strictly controlling the nature o f costimulation by CD28 but not CTAL-4, such approaches may avoid natural down-regulatory signals like CTLA- 4. Costimulation ex vivo through CD28 results in the generation o f tumor-specific CTLs. This approach has been effective in experimental models o f established metastatic tumors (Renner et al.. 1996). The second approach is to systemically enhance costimulation in vivo at the T cell level, typically involving intravenous administration o f agents that are targeted to the T cells. Specific targeted approaches have used bispecific antibodies such as anti-CD3/antitumor antigen to generate signal one plus monospecific anti-CD28 monoclonal antibody, antitumor antigen/anti-CD28 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. bispecific monoclonal antibody to enhance costimulation, or anti-CD3/antitumor antigen plus antitumor antigen/anti-CD28 bispecific monoclonal antibodies to augment both signal one and signal two. Such approaches eliminate established tumors and enhance the immunogenicity o f tumor cell vaccines in vivo (Renner et al., 1994). Conversely, another approach is to block CTLA-4 receptor activation with monoclonal antibodies to prevent down-regulation of T cell activation (Leach et al., 1996). This approach has resulted in complete regression of established unmodified tumors in experimental murine models of fibrosarcoma, colon carcinoma, and prostate carcinoma. A third approach is to turn tumor cells into APCs by gracing them with the ability to express costimulatory ligands. This is essentially a whole-cell vaccination approach. It has been demonstrated that membrane incorporation of giycosyl-phosphatidyinositol (GPI)-anchored CD80. or transfection o f CD80 or CD8 6 genes into hematopoietic and non-hematopoietic tumor cells leads to a robust antitum or response when transfected cells are implanted into syngeneic host animals (Matulonis et al., 1996: McHugh et al.. 1995). These responses are mediated by CD8 T cells, indicating that B7 transfected tumors likely serve to activate and expand antigen-specific CD8^ CTL which then are capable of killing both transfected and parental cells. Another strategy is the vaccination with tumor antigens and costimulatory ligands. The genes for costimulatory ligands together with antigenic genes are delivered to the target cells, providing both signal one and two to the T cells. For 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancer immunotherapy, such “intracellular” immunization mimics viral infection w ith the added advantage of inducing vigorous cell mediated immune responses via endogenous antigen presentation by MHC I to reactive CD8 ^ CTLs. Immunization with a recombinant adenovirus, which infects a wide variety o f cell types that is designed to encode hepatitis B surface antigen and CD80. leads to more robust antibody and CTL responses (He et al., 1996). Immunization with mixtures of recombinant vaccinia viruses encoding human carcinoembrvonic antigen and CD80 results in enhanced antitumor responses (Hodge et al.. 1995). 1.8. Scope of the dissertation Because of the importance of costimulatory molecules in the control o f immune responses as described above, understanding their expression and regulation on antigen presenting cells will provide us the information that is needed to modulate an immune response. We are especially interested in the B7 expression on macrophages due to their unique heterogeneity in function, when compared with other two types o f antigen presenting cells. B cells and dendritic cells. The research in this dissertation composes four parts: 1. Comparison of B7 expression on macrophage subpopulations (Chapter 2). Macrophages are a very heterogeneous population in terms of distribution and function. The B7 expression and TNF-a production in resident peritoneal 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. macrophages, thioglycollate elicited peritoneal macrophages and alveolar macrophages were compared. 2. Can crosslinking o f CD40 upregulate B7 expression on peritoneal macrophages (Chapter 3)? It has been demonstrated that the interaction between CD40 and CD40L on B cells and T cells, respectively, can help the increased B7 expression on B cells. Using CD40L transfected cells or activated T cell membrane has been shown to increase B7 in DCs. Immobilized anti-CD40 antibody was used to examine the effect of crosslinking CD40 on B7 expression in macrophages in this study. 3. Quantitation o f B7 molecules (Chapter 4). It is well established that B7 is important for T cell activation. However, how many molecules are necessary to trigger the response is not known. The number o f B7 molecules on each macrophage was determined using standard beads. Together with the proliferation assay, the number o f B7 molecules required for T cell proliferation was determined. 4. Development o f ELISA to measure B7 (Chapter 5). B7 is measured with FACS in almost all studies described in the literature. Although it is sensitive and quantitative, auto fluorescence is a major problem. In the case o f measuring B7 on macrophages, the cells have to be removed from the 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. culture plate by trypsinization. a procedure that may destroy the cell surface molecules o f interest. An alternative method to measure cell surface molecules is enzyme-linked immunosorbent assay (ELISA). The F(ab')2 fragment o f anti-B7- 2 antibody G LI was directly coupled to P-galactosidase using a novel heterobifunctional crosslinker Sulfo-SMCC. This reagent was used in one-step ELISA to measure B7-2 expression on B cells and a comparison was made with that measured using FACS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER TWO COMPARISON OF B7 EXPRESSION ON MACROPHAGE SUBPOPULATIONS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.1. Introduction Macrophages are a heterogeneous population of cells differing in their location, morphology and function. They develop from hematopoietic stem cells originating in both fetal liver and bone marrow. Multiple mechanisms have been proposed to underlie the development o f functionally heterogenous macrophage populations. First, differential signals may be experienced within diverse tissue microenvironments (Andreesen et al., 1990). It has been long recognized that macrophages isolated from different anatomical sites display a diversity of phenotypes and capabilities. Because macrophage function is dependent in part on signals received from the immediate microenvironment, it has been suggested that macrophage heterogeneity may arise from the unique conditions within specific tissues. In fact, each tissue determines the functional phenotype of its resident macrophage population (Damoiseaux et al.. 1989). Secondly, macrophage heterogeneity is associated with maturation stage. Macrophage differentiation is accompanied by the transient expression of defined phenotypes (Neumann and Sorg, 1980). The third factor that causes the macrophage heterogeneity is the clonal variation o f myeloid progenitors. It has been suggested that heterogeneity exists prior to release of the monocyte from the marrow, perhaps due to the existence o f distinct progenitor cells. Bone marrow progenitor cells are heterogeneous in size and proliferative capacity, even for clones that yield one morphological cell type (Metcalf, 1989). Macrophage 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. progenitor cells in peripheral tissues may constitute an additional lineage system. The fourth factor responsible for the macrophage heterogeneity is alternate hematopoietic stimulation. In vitro, the growth factor with which they are propagated, the period o f differentiation, and the addition o f exogenous stimuli may result in different phenotypes (Witsell and Schook. 1991). The in vivo differentiation o f macrophage populations is definitely more complex. Progenitor cells experience combinations of factors with both positive and negative differentiation effects, especially during an inflammatory response. The functional plasticity o f macrophages gives the immune system added flexibility to respond to challenge. It is likely that the nature o f an immune response is dictated in large part by the functional phenotypes o f the macrophages present within the lesion. Distinct Th cell subsets such as Thl or Th2 cells may favor the production or activation of a particular macrophage subset (Street and Mosmann, 1991). On the other hand, heterogenous macrophage populations may differentially regulate or favor the production o f distinct Th cell populations, thereby altering inflammatory responses (Gajewski et al.. 1991). Among the signals that activate macrophages is lipopolysaccharide (LPS). LPS or endotoxin, a constituent of the outer cell wall of Gram-negative bacteria, consists of four structurally and functionally distinct regions as illustrated in Scheme 2.1: the O-specific chain, the outer core, the conserved inner core oligosaccharide, and the lipophilic lipid A moiety (Rietschel et al.. 1982). The O- specific chain is characteristic for a given LPS and varies between species and 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Abe ; (Gal-Rha— Mari)n I Glc II — GlcNAc Hep II— Hep III KDO I - KDO II—(KDO III) O >= O ■ = O — a = o >=0 I O-Specific chain Outer core Inner core Lipid A Scheme 2.1. Structure o f LPS o f Salmonella entericci. GIcN. glucosamine; GlcNac, N-acetylglucosamine; KDO. 2-keto-3-deoxyoctulosonic acid; Hep. L- glyco-D-mannoheptose; Glc, D-glucose; Gal, D-galactose; Rha. L-rhamnose; Man, D-mannose; Abe, Abelose. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. serotypes of bacteria. The inner cores o f a variety of LPS are chemically similar and contain at least one 2-keto-3-deoxyoctulosonic acid (KDO) residue or a KDO derivative. Most LPS inner cores contain heptose residues. The lipid A moiety is thought to anchor the LPS onto the bacterial outer cell wall, and hence lysis of the bacterial wall is required for exposure o f lipid A to other binding proteins. Lipid A contains a P (l-6)-linked disaccharide with one glycosidic and one non- glycosidic phosphate group and medium- to long-chained (CIO to C28) fatty acids linked by amide and ester bonds. The full biological activity o f LPS requires two hexosamine residues, two phosphoryl groups, and six fatty acids (Rietschel et al.. 1990). Cellular recognition of LPS involves several different molecules and the most important one is cluster of differentiation antigen 14 (CD 14). CD 14 is a 55 kDa glycoprotein anchored to the cell surface via a phosphatidyl inositol anchor (PI) that does not cross the cell membrane and leaves it mobile within the plane of the membrane (Haziot et al., 1988; Wright et al.. 1990). CD14 is expressed on the surface o f monocytes, macrophages, and neutrophils. CD 14 expression has been reported to be influenced by a variety o f factors including in vitro monocyte to macrophage maturation, various cytokines, and LPS itself. In addition to the membrane form o f CD14, a soluble form o f CD14 (sCD14) is found in serum. Its molecular weight is about 50-53 kDa as estimated by SDS-PAGE (Bazil et al.. 1986). sCD14 is believed to originate from enzymatic cleavage of membrane CD 14 via the action of specific phospholipases or proteases (Bazil et al.. 1989). 37 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sCD14 binds to LPS and prevents its subsequent interaction with cellular LPS receptors. More importantly, sCD14 may serve to activate CD 14-deficient cells in response to LPS (Lielian and Blecha, 1995). Another essential component involved in the interaction between LPS and CD 14 is a 60 kDa serum glycoprotein lipopolysaccharide binding protein (LBP). LBP binds with high affinity to the lipid A portion of LPS and then interacts with CD 14 (Tobias et al.. 1989). The amino terminal half o f the protein is responsible for the specific binding to LPS while the carboxyl-terminal half o f LBP is responsible for CD 14 interaction. LBP facilitates the interaction o f LPS with CD 14 by two basic processes. First, LBP acts as an opsonin for LPS-bearing particulates enhancing the interaction with CD 14 (Wright et al.. 1989). and secondly. LBP enables cells to respond to extremely low concentrations o f LPS via a CD 14-dependent pathway (Schumann et al.. 1990). The propagation o f the LPS signal from the plasma membrane to the nucleus is complex. It involves protein tyrosine kinases, mitogen-activated protein kinases, protein kinase C. G proteins, protein kinase A. ceramide-activated protein kinase, and microtubules. At the nuclear level rel. C/EBP. Ets. Egr. fos. and jun family members have been implicated in activation of LPS-inducible gene expression (Sweet and Hume, 1996). Although CD 14 has been shown to be an important effector molecule in LPS-mediated macrophage activation, evidence exists for alternate receptors for LPS. One of the well-characterized receptors displaying affinity for LPS is the 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. leukocyte integrin CD 18, including all three members o f the family. CD 11 a/CD 18. CD1 lb/CD 18 and CD 11 c/CD 18 (Wright and Jong. 1986). CD 18 can recognize LPS and LPS in association with whole bacteria. Other LPS receptors are 73 kDa, 38 kDa and 55 kDa cell surface proteins (Lielian and Blecha, 1995). In this study three types o f macrophage subpopulations, resident peritoneal macrophages, thioglycollate elicited peritoneal macrophages and alveolar macrophages, were compared, especially their reactivity to LPS, using expression o f B7 as an indicator, since costimulation is a critical function of macrophages. Injection o f thioglycollate into the peritoneal cavity induces a sterile inflammatory reaction and recruits blood monocyte-derived macrophages into the peritoneal cavity. Alveolar macrophages (AM) represent 85% o f the cells retrieved by bronchoalveolar lavage. They are bone marrow-derived cells that can be differentiated from blood monocytes after they have emigrated into the tissues. AM are the resident phagocytic cells in the lung and constitute the first line of defense against infection agents that reach the gas-exchanging airway. In addition to its phagocytic capability, the AM plays a prominent role in controlling the inflammatory and immune response by releasing cytokines, chemokines. reactive oxygen or nitrogen intermediators and complement proteins. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2. Materials and Methods 2.2.1. Animals BALB/c mice, 8 to 12 weeks of age, were purchased from the Jackson Laboratory (Bar Harbor, ME) and were maintained in the animal facilities at the University o f Southern California in accordance with institutional guidelines for animal care. 2.2.2. Cell culture medium The tissue culture medium (TCM) used for cell culture was based on Click's medium (Irvine Scientific, Santa Ana. CA) which was supplemented with 100 U/ml penicillin, 100 p.g/ml streptomycin. 4 mM L-glutamine (all from Life Technologies, Gaithersburg, MD), 40 p.M p-mercaptoethanol (Sigma), and 10% fetal bovine serum (FBS. HyClone. Logan, UT). 2.2.3. Reagents Sodium chloride, potassium chloride, sodium phosphate (monobasic, monohydrate), and sodium phosphate (dibasic, heptahydrate) used for preparing PBS, heparin. LPS, sodium azide, citric acid, Tween-20, 30% I-LCL. O- phenylenediamine (OPD), and avidin-horseradish perodixase (HRP) were from Sigma. Trypsin-EDTA (10X) was purchased from Life Technologies. Nembutal® 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sodium solution (pentobarbital sodium injection) was the product o f Abbott Laboratories. Rat anti-mouse TNF-a, hamster anti-mouse CD40 (HM40-3). and biotin rabbit anti-mouse TN F-a were purchased from PharMingen. F(abr)2 fragments o f monoclonal antibodies (mAbs) 1G10 (anti-B7-l) and GL1 (anti-B7- 2) were generated by digestion o f purified antibodies by pepsin. FITC labeled goat F(ab')2 anti-rat IgG was from Southern Biotechnology. 2.2.4. Preparation o f macrophages For the preparation o f resident peritoneal macrophages, the mice were sacrificed by cervical dislocation. The peritoneal cavity was washed with 10 ml cold sterile PBS containing 20 U/ml heparin and again with another 5 ml fresh solution. The solution containing the peritoneal exudate cells (PECs) was pooled and spun down. The cells were then resuspended in TCM with 10% FBS. For thioglycollate elicited peritoneal macrophages, the cells were harvested 4 days after intraperitoneal injection of 1.5 ml 6% thioglycollate medium. Alveolar macrophages were obtained by tracheobronchial lavage. The mice were anaesthetized by Nembutal® sodium solution, injected intraperitoneallv and the lungs were washed by 10 consecutive 1ml cold PBS containing 20 U/ml heparin in situ. The cells were washed and re-suspended in TCM containing 10% FBS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For purification, the PECs and trachobronchial lavage cells were plated into wells o f 6-well or 24-well plate, incubated for 2 h at 37°C in a humidified atmosphere o f 5% CO2 in air. Nonadherent cells were removed by gentle wash three times with TCM. More than 90% o f the adherent cells from the peritoneal cavity were macrophages as defined by positive staining for Mac-1. More than 90% o f adherent cells from lung were macrophages as determined by their forward and side scatter characteristics. 2.2.5. FACS analysis o f B7-1 and B7-2 The macrophages were removed from the tissue culture plate by treating them with I OX trypsin-EDTA and transferred to round bottom multiwell plates ( I -2x 103 cells/well). After washing once with staining buffer, the cells were incubated with 50 pi o f 10 pg/m l o f anti-B7-l antibody (F fab 'h fragment o f 1G10) or anti-B7-2 antibody (F(ab')2 fragment of GL1) for 30 minutes on ice. washed three times with staining buffer, stained with 50 pi FITC-labeled second step antibody, i.e. FITC goat F(ab')2 anti-rat IgG. for 30 min on ice in dark, and washed again three times. For each sample. 5x10J viable cells were analyzed on a FACScan using CellQuest software (Becton Dickinson. Mountain View. CA). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.6. ELISA for TN F-a Purified rat anti-mouse TNF-a antibody, diluted in 0.1 M NaiHPCL (pH 9.0) to a concentration o f 2 pg/ml (50 pi), was added to each well of an Immunlon 4 ELISA plate (Dynatech Laboratories). The plate was sealed and incubated overnight at 4°C. After washing twice with PBS/Tween (PBS containing 0.05% Tween-20), the non-specific binding sites o f the plate were blocked by incubating it with 200 pi PBS/10% FBS at room temperature for 2 h. The plate was washed again 3 times with PBS/Tween, followed by addition o f 50 pi recombinant TNF- a standards and macrophage culture supernatant diluted in TCM to different wells o f the plate. The plate was sealed and incubated at 4°C overnight. The plate was then washed 4 times and 50 pi of 1 pg/ml biotinylated rabbit anti-TNF-a detecting antibody in PBS/10% FBS was added to each well. The plate was incubated at room temperature for 1 h. and then washed with PBS/Tween 6 times. Avidin-HRP (50 pi o f 2.5 pg/ml diluted in PBS/10% FBS) was added to the plate, followed by incubation at room temperature for 30 min. and washed 8 times with PBS/Tween. HRP activity was measured by adding its substrate O- phenylenediamine (OPD). Tablets containing OPD were dissolved in 0.05 M phosphate-citrate buffer (pH 5.0) to a final concentration o f 0.4 mg/ml and 40 pi o f 30% H2O2 was added to 100 ml substrate solution immediately prior to use. The substrate solution (50 pi) was added to each well, incubated at room temperature in the dark for 30 min to 1 h. Depending on the rate of color 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. development, the reaction was stopped by adding 50 pi 3 N HC1 and the absorbance was read at 490 run. 2.3. Results 2.3.1. Comparison o f B7 expression on alveolar macrophages (AM), resident peritoneal macrophages (rPM) and thioglycollate elicited peritoneal macrophages (PM) The B7 expression on ex vivo macrophages, macrophages cultured in TCM alone, and macrophages stimulated with 10 pg/ml LPS was measured by FACS. As shown in Figure 2.1, the AM had the lowest level o f B7-1 and B7-2 under all conditions, LPS could increase the B7-2 expression, but not to a level similar to that on peritoneal macrophages. The ex vivo rPM and PM showed a substantial and similar amount of B7-2 expression, indicating that peritoneal macrophages constitutively express B7 and thioglycollate did not change this expression. TCM had little effect on B7 expression on these two populations of macrophages. However, these two populations responded to LPS to a different extent. B7-2 on PM was increased more than on rPM. 2.3.2. Low expression of B7 on alveolar macrophages (AM) Since a significant effect o f LPS on B7 expression was not observed in AM. a combination o f several stimuli was tested as shown in Figure 2.2. Crosslinking 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. of CD40 has been shown to be able to increase B7 on APCs. In our experiment this is achieved by using immobilized anti-CD40 antibody. IFN-y is another activator o f macrophages and it has been proposed that macrophages need to be primed by IFN-y before they could react to LPS. Therefore, the combinations of LPS and crosslinking of CD40. IFN-y and crosslinking o f CD40. LPS plus IFN-y plus crosslinking o f CD40 were used to stimulate AM. Although the combined stimuli had some additive effect, the level o f B7-2 expression was still lower in AM than in rPM and PM. The comparison o f B7 on AM and PM was done after 24 h of stimulation. At this time point the B7 expression on PM has reached its peak as illustrated in Figure 2.3. the time course of B7 expression. Although B7 expression on AM may follow different kinetics, at least at the 24 h point. B7 expression is quite different between AM and PM. 2.3.3. TN F-a production by macrophage subpopulations Production o f TN F-a is another major function of activated macrophages. In contrast to B7 expression. AM released more them 30 times more TN F-a than PM upon stimulation with LPS (Figure 2.4). A small am ount o f TN F-a was measured from AM after crosslinking CD40. This result demonstrated that macrophage functions o f each subpopulation are differentially regulated by LPS. No detectable amount o f TN F-a was produced by resident peritoneal macrophages, even in the presence o f LPS. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 100 AM C I $ ® 0 ■ ; I ! ^ 60 i < U | O ; S 40 j co ! ( D i ° on ! 3 2 0 -i U _ TJ CO -20 rPM 1 1 1 J. o o 13 TJ C O M (D X < <’ o PM o C O ■ B 7-1 0B7-2 Figure 2.1. Comparison o f B7 expression on different macrophage subpopulations. Alveolar macrophages (AM), resident peritoneal macrophages (rPM) and thioglycollate elicited peritoneal macrophages (PM) were prepared as described in Materials and Methods. The cells were cultured in the absence (control) or in the presence o f 10 pg/ml LPS for 24 hours and the B7 was measured by FACS. 46 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 2.2. Expression o f B7 on alveolar macrophages. Alveolar macrophages were obtained from BALB/c mice as described in Materials and Methods. 10 pg/ml of anti-CD40 was immobilized on a 24-well plate. LPS was used at 10 pg/ml and INF-y at 100 U/ml. After 24 h o f culPire with the reagents as indicated. B7 expression on alveolar macrophages was measured by FACS. 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24 h 48 h 72 h 96 h 300 200 - < D 150 - g 100 o o i " O C O "0 C O o o Z J " O C O ■ B7-1 E3 B7-2 Figure 2.3. Time course o f B7 expression on thioglycollate elicited peritoneal macrophages (PM). PM were cultured in the tissue medium alone or in the presence of 10 pg/m l LPS at the density o f 106 cells/ml. Cells were collected at different time points and the B7 expression was measured by FACS. 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 200 -| AM/LPS AM/CD40 PM/LPS PM/TCM rPM/LPS Figure 2.4. TNF-a production by macrophage subpopulations. Different macrophage subpopulations were cultured in the 6-well plate with TCM alone, in the presence of 100 pg/ml LPS or wells coated with 10 pg/ml anti-CD40. 24 h later the supernatant was collected and the amount o f TN F-a was measured by ELISA. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.4. Discussion The observed different sensitivity o f B7 expression in AM and PM to LPS may be caused by the various levels of CD 14 expression, the receptor for LPS. A heterogenous expression of CD 14 on mature tissue macrophages is dependent on the tissue of origin. It has been reported that human alveolar macrophages express low levels of CD 14. whereas peritoneal macrophages possess much higher levels of membrane CD14 (Andreesen et al.. 1990). This CD14 expression pattern m ay also hold true in mice. The reduced B7 expression in AM may be advantageous, as a critical mechanism that limits immune responses in the lung and in mucosal tissues which are continuously exposed to abundant quantities of antigens. Under normal conditions, despite the expression o f normal levels o f MHC II antigens and having normal bactericidal activity. AM have been shown to be defective in activating resting T cells and in initiating inflammatory responses (Chelen et al.. 1995). However, in pathologic states, such as sarcoidosis. AM can express functional costimulatory molecules. like CD86. for T cell activation (Nicod and Isler, 1997). The results presented here indicate that B7 expression is regulated differently in AM than in PM, suggesting that the role o f AM in immune responses is distinct from that o f other types of APCs. AM may be involved in the induction of peripheral tolerance rather than the activation of T cells. The 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reduced expression o f B7 in AM may play a major role in downregulating immune responses in the lung and in inducing immunological tolerance to inhaled antigens. But how are antigen-specific immune responses elicited in the lung? Pulmonary dendritic cells (DC) have been considered to be the most potent antigen presenting cells in the lung. Unlike AM. DCs are present in relatively small numbers but are widely distributed in the normal lung, including the pleura, alveolar septal interstitium, pulmonary capillaries, peribronchiolar connective tissue and bronchus associated lymphoid tissue (Hance. 1993). Lung DC. like DC from many sources, have the remarkable ability to stimulate lymphocyte proliferation. Lung DCs are generally 10-100 fold better than AM in inducing lymphocyte proliferation. AM may actually suppress the lymphocyte proliferation induced by DC (Hance, 1993). Another dramatic difference between AM and PM is the amount o f TN F-a production after LPS stimulation. The higher level o f TNF-a produced by AM will recruit other effector molecules to the site o f infection, and therefore effectively fight with infectious antigens and microorganisms that AM frequently encounter. Thioglycollate has been claimed to enhance some macrophage functions but does not fully activate the cells (Unanue and Allen. 1987). This is confirmed by our data. The thioglycollate PM consistently express higher level o f B7 than resident PM in response to LPS. Whether it is through the upregulation o f CD 14 or other mechanisms is not known. The higher level of B7 expression in PM. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. compared with in AM, implies that PM are more efficient in inducing an antigen specific immune response against invading antigens than AM. 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER THREE REGULATION OF B7 BY SIGNALS FROM T CELLS Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.1. Introduction Cell communication is essential for maintaining homeostasis, and for the ability to respond to environmental insults such as infections. Hence, within the immune system, complex regulatory networks exist that include both communication via soluble mediators, like cytokines, and direct cellular contacts via specific surface receptors. For example, the development o f an efficient immune response is dependent on the receptor ligand pair o f CD28-B7 as mentioned before. Another receptor ligand pair involved in the generation o f immune responses that has recently been extensively studied is CD40 and its ligand CD40L. CD40 is a 50 kDa member o f the tum or necrosis factor receptor (TNFR) family, which includes TNF-RI, TNF-RE, Fas, CD30, CD27, 4-1BB, and OX-40 (Baker and Reddy, 1996). CD40 expression has been described on all antigen presenting cell types, including B cells, dendritic cells, and monocytes/macrophages. Stromal cells that express CD40 include thymic epithelia, vascular endothelium, keratinocytes, and fibroblasts. CD40 expression is constitutive on B cells and can be further up-regulated by IFN-y and GM-CSF (Stamenkovic et al., 1989). Its expression is also constitutive on cultured and freshly isolated dendritic Langerhans cells. Freshly isolated splenic DCs express low levels o f CD40 but up-regulate its expression after adherence in vitro (Alderson et al., 1993). 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CD 154 (CD40L, gp39) is a 32 to 39 kDa member o f the TNF family, which includes TNF-a. LTa. LTp, FasL, CD30L. CD27L. 4-1BBL and OX-40L (Baker. Reddy, 1996). Activated CD4+ T cells are the predominant cell type expressing CD 154. Expression on CD8+ T cells, eosinophils, mast cells and basophils, natural killer (NK) cells, and DCs has also been described (Roy et al.. 1993). Expression of CD154 after activation o f CD4+ T cells with anti-CD3 is observed beginning at 4 h. peaks between 6 and 8 h and is down-regulated by 24 h. CD 154 is induced by anti-CD3 more rapidly and is expressed at higher levels on effector than naive CD4’ ’ ’ T cells, which may account for the reduced costimulatory requirements o f memory T cells for their activation following subsequent exposure to antigen (Dubey et al.. 1996). Studies on CD40 signal transduction in B cells have now resulted in a more precise picture o f the different mediators and pathways involved in this process. Alternative pathways may be operational in other cells types. Like all other members o f the TNFR family, CD40 has no kinase domain and no known consensus sequence for binding to kinases, yet, CD40 ligation activates several second messenger systems. including protein-tvrosine kinases, phosphatidylinositol 3-kinase, phospholipase Cy2 as well as the serine-threonine stress-activated protein kinases. These different activation pathways ultimately result in the activation o f various transcription factors including NF-kB. N F-kB- like molecules, c-jun and NF-AT (FCehry, 1996). The direct coupling o f the CD40 55 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. receptor to different signaling pathways has been further explored by the identification o f a new family o f associated proteins: TNFR associated factors (TRAF). Using the two-hybrid system, such protein-protein interactions have been demonstrated for different members of the TNFR family. Interaction o f CD40 with CD40L results in a multimerization o f the CD40 complex. CD40 can be associated with various TRAF molecules. These TRAF molecules have additional domains for protein-protein interaction, and as a result o f the physical aggregation, will form homo- and heterodimeric complexes. As a consequence, further downstream signal transduction can be initiated (Kehry. 1996). In vitro studies have demonstrated that CD40 activation has major effects on many steps o f the B cell natural history: proliferation o f immature and mature B cell subsets, differentiation and Ig production, isotype switching, rescue from apoptosis, phenotypic differentiation into germinal center cells and skewing o f the maturation o f germinal center cells into memory cells rather than plasma cells (Van Kooten and Banchereau. 1996). CD40 ligation on monocytes and dendritic cells results in an enhanced survival o f these cells, the secretion o f cytokines (such as IL-1, IL-6, IL-8 1L-10. IL-12. TNF-a. M IP-la) and enzymes, enhanced monocyte tumoricidal activity and NO synthesis (Stout and Suttles, 1996). The role o f CD40-CD40L interactions in T cell responses was investigated by using CD40L knock-out mice (Grewal et al., 1995). The CD40LT mice are severely impaired in primary T-cell responses to protein antigens. The defective T cell responses o f these mice were mapped to an inability o f CD40L-deficient T 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells to undergo effective clonal expansion in vivo and to enter the cell cycle. An adoptive transfer system was used to monitor the expansion o f a limited number o f T cells from TCR transgenic mice following their transfer into normal recipients that were challenged with antigen. T cells lacking CD40L failed to expand in vivo, whereas wild-type cells expanded normally, indicating that CD40-CD40L interaction is required for effective in vivo priming o f CD4^ T cells. Two possible explanations of the impairment o f T cell mediated immune responses in CD40L deficient mice can be considered. The first possibility is a lack o f a direct signal transduced to T cells via CD40L itself, which may be responsible for the impairment o f cell mediated immunity. A second possibility is that an indirect signal is mediated by CD40-CD40L interactions for the activation/priming of APC, which is required for T cell activation and the development o f effector functions. Evidence is accumulating favoring the second possibility suggesting that CD40-CD40L interactions play a critical role in the initiation o f a T cell response by up-regulating costimulatory molecules on APCs such as B cells and dendritic cells (Caux et al., 1994). Interactions between T cells and APCs must be carefully regulated to prevent the activation o f unwanted self-reactive or bystander T cells. The presence o f CD40 and MHC molecules on resting APCs. and TCR and CD28 on resting T cells, is not sufficient to trigger an immune response. A reciprocal dialogue between APCs and T cells can only occur if either APCs or T cells are 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activated. A two-step model was proposed to describe how APCs and T cells communicate with each other leading to the activation o f T cells and the initiation o f the immune response as shown in Scheme 3.1 (Grewal and Flavell. 1996) based on the studies of B cells and dendritic cells. During the first step, the T cells see antigen on the APCs. which in most cases will be dendritic cells. The T cells receive the antigen signal (signal one), together with additional signals provided by APCs, such as cytokines or adhesion molecules, which cause the up- regulation o f CD40L. This in turn activates the expression o f the costimulatory activity o f the dendritic cells and other APCs. In the second phase o f T cell activation, the costimulatory signal is received by the T cell via CD28. which drives the T cells into proliferation and cytokine production. This model provides an additional regulatory step in the initiation of the immune response, that is. the activation o f the APCs primed with the cognate antigen by the T cell specific to that antigen. Thus, this step, coupling the activation o f T cells specific for a given antigen and the APCs carrying that antigen, may provide a means for prevention o f an autoimmune response by not activating bystander self-reactive T cells during the initiation of an immune response. In this model the mechanism o f inducing costimulatory molecule expression is quite different from the model proposed by Charles Janeway that APCs carry out self-nonself discrimination and convey the result to T cells via costimulation (Janeway. 1992). In the present study, the effect o f signals from T cells on B7 expression on thioglycollate elicited peritoneal macrophages was investigated. The interaction 58 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. APC T cell Peptide Antigen .MHC TCR ‘CD40 CD28 CD40L ‘CD40 Activation B7 Signal 2 Scheme 3.1. The role o f CD40-CD40L in T cell activation. A two-step model of activation of T cells. In the first step antigens are taken up by naive APCs (e.g. unprimed DC), and present processed antigen to naive T cells in the form of MHC/peptide complexes, which deliver the antigenic signal (signal one) via TCR to naive T cells. As a result, T cells up-regulate CD40L on their surface. In step two, CD40L on the surface o f T cells induces costimulatory activity on DC via the CD40-CD40L interaction. This primed APC now expressing costimulatory molecules sends a second costimulatory signal to T cells alone with an antigen signal for the full activation of T cells to produce cytokines and to perform effector functions. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o f CD40 on macrophages and CD40L on T cells was mimicked and simplified by adding macrophages to immobilized anti-CD40 antibody. The regulatory effect o f some soluble mediators on B7 expression, like IFN-y. GM-CSF. and PGE2- was also investigated. 3.2. Materials and Methods 3.2.1. Animals, cell culture medium, macrophage preparation. FACS analysis of B7-1 and B7-2 were described in 2.2.1, 2.2.2. 2.2.4. and 2.2.5. respectively. 3.2.2. Preparation of B cells. B cells were prepared from spleen cells. BALB/c mice were sacrificed by cervical dislocation. The spleen was excised and a single cell suspension was prepared. The red blood cells were removed by hypotonic lysis with water. T cells were removed by incubating the cells with 10 f-ig/ml purified anti-Thyl.2 antibody (30-H12) at a density of 107 cells/ml on ice for 30 min. followed by incubation with 1:10 rabbit complement (ICN Biomedicals) at the same cell density at 37°C for 45 min, shaking every 10 min. The purity was above 95% as determined by staining with anti-B220 antibody using FACS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2.3. Measurement o f CD40 Macrophages were incubated with 20 pg/ml. 50 (.d/well anti-FcR monoclonal antibody 2.4G2 on ice for 30 min to block Fc receptors. After washing with staining buffer, the cells were incubated with I f-ig/ml primary antibody, hamster anti-mouse CD40 (HM 40-3. IgM). on ice for 30 min. For detection, the cells were first incubated with 2 jig/ml biotinylated mouse anti hamster IgM (G188-9) for 30 min., washed, followed by the addition o f avidin conjugated to phycoerythrin. After 30 min on ice. the cells were washed and CD40 expression was measured by FACS. 3.3. Results 3.3.1. Effect o f CD40 crosslinking on B7 expression. Up-regulation o f B7 on human monocytes has been observed when they were cultured together with CD40L transfected cells or membranes prepared from activated CD4T T cells (Wagner et al.. 1994). In this study, immobilized CD40 antibody was used as a substitute approach to achieve the crosslinking o f CD40 on peritoneal macrophages. 0.1, 1. and 10 pig/ml purified anti-CD40 antibody was precoated on the 24-well plate. Macrophages or B cells (106) were added to the plate, cultured for 18 h and the B7 expression was measured by FACS. As shown in Figure 3.1, no elevated B7 levels were detected compared with control. 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. on macrophages cultured in anti-CD40 coated plates. This was not due to a defect in the macrophages, because they could respond to LPS and display enhanced B7- 2 expression. On the other hand, the failure o f crosslinking o f CD40 by immobilized anti-CD40 could not explain the result. As the positive control, in B cells, crosslinking of CD40 had the similar effect as LPS in enhancing the B7-2 expression, although the level was lower than that in macrophages. To further confirm the result, a time-course experiment was performed. In Figure 3.2, the increased B7-2 expression in B cells lasted for 72 h. However, no increased B7-2 expression in macrophages was observed during this time period. Another possibility was that the crosslinking of CD40 was not achieved because of low density of CD40 on macrophages. Therefore, the macrophages were cultured with IFN-y, which is known to up-regulate CD40. The B7-2 expression in macrophages cultured in the presence of IFN-y alone or together with the immobilized CD40 was compared in Figure 3.3. It is clear that despite the increased CD40 level. B7-2 was not further increased by the crosslinking of CD40. Collectively, crosslinking of CD40 in macrophages with immobilized anti-CD40 could not increase B7 expression in these cells. 3.3.2. Soluble effectors on B7-2 expression in macrophages. IFN-y is produced by T hl CD4 T cells and CD8 T cells. It is an important and multifunctional mediator in the immune system. It has been known that INF- y is an activator of macrophages. The specific effect o f INF-y on the B7-2 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression in macrophages was studied. Figure 3.4 shows that when the concentration o f IFN-y was higher than 10 U/ml. it had an effect similar to that of LPS in up-regulating B7-2. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is another major cytokine released from CD4 T cells acting on bone marrow cells to promote the maturation of macrophages from their progenitors. The direct effect of GM-CSF on B7 expression in macrophages was investigated. The results in Figure 3.5 demonstrate that GM-CSF had no effect on B7 expression on mature macrophages. Thus, the function of GM-CSF may be more important in differentiation, proliferation o f macrophages in their maturation process rather than in their effector phase. Another soluble factor studied was prostaglandin Ei (PGE2). PGE2 is the product o f macrophages themselves. It has been shown that PGE2 is a negative modulator o f B7 expression and it can abolish the effect of LPS in microglia, the macrophage population in the brain. Its effect on peritoneal macrophages was examined and the result is shown in Figure 3.6. In this experiment. PGE2 had no effect on the effectiveness o f LPS in the up-regulation of B7. The opposite effects o f PGE2 on microglia and peritoneal macrophages may again reflect the heterogeneity o f macrophage subpopulations. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 300 B Cells Macrophages c n C D C D O C D O C/5 2 O 3 200 - 100 - o > > X 0 ) -100 J o O ■I C O CL O Q O c C D > X a > i i co ^ Q_ o O O ■ B7-1 0 B7-2 1 anti-CD40 (ug/ml) Figure 3.1. Differential effect o f crosslinking CD40 on B7 expression in B cells and thioglycollate elicited peritoneal macrophages. The 24-well plate was coated with 10 pg/ml or indicated concentrations of monoclonal anti-CD40 antibody in PBS at 4°C overnight. After washing out the unbound antibody. 10& B cells or peritoneal macrophages were added to the plate. LPS was used at 10 pg/ml. Cells cultured in tissue culture medium alone were used as control. 18 h after culture the cells were collected and B7 was measured by FACS. 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. B Cells Macrophages □ Control 0LPS ■ Anti-CD40 24 48 72 24 48 72 Time (h) Figure 3.2. Time course o f B7-2 expression in B cells and thio glycol late elicited peritoneal macrophages after crosslinking of CD40. The 24-well plate was coated with anti-CD40 as described in Figure 3.1. Cells were cultured in wells with tissue culture medium alone (control), in the presence o f 10 pg/ml LPS, or wells with 10 p.g/ml immobilized anti-CD40. Cells were collected at different time points and the level of B7-2 was measured by FACS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 120 100 c ra | 80 E 2 B7-2 □ CD40 < D £ 60 0 ) o c n < D o 40 Z3 U _ 20 anti-CD40 + IFN -/ Figure 3.3. Increased CD40 expression on macrophages did not enhance the B7-2 expression. A 24-well plate was coated with 10 |-ig/ml anti-CD40 antibody. 106 peritoneal macrophages were added to a well containing 100 U/ml IFN-y or to the anti-CD40 coated well together with 100 U/ml IFN-y. The cells were collected after 24 h culture and the expression of B7-2 and CD40 was measured by FACS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 180 -i 160 - ex vivo 0 0.1 1 10 100 LPS INF-y (U/ml) Figure 3.4. The effect of IFN-y on B7-2 expression in thioglvcollate elicited peritoneal macrophages. 106 cells were cultured in the tissue culture medium alone, in the presence o f indicated concentrations o f INF-y. or with 10 p.g/ml LPS. The cells were collected 18 h after culture and the B7-2 expression was measured by FACS. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. QB7-2 ® 30 - ex vivo GM -CSF (ng/mi) Figure 3.5. The effect o f GM-CSF on B7 expression in thiogiycollate elicited peritoneal macrophages. 106 cells were cultured in the tissue culture medium alone, or in the presence o f indicated concentrations o f GM-CSF. The cells were collected 18 h after culture and the B7 expression was measured by FACS. 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 140 i — 120 - C O I 100 aj 80 0 1 60 C /> ( 1 ) 5 40 20 - 1 m i ■ B 7-1 SB7-2 Ex vivo Control L PS LPS LPS LPS PGEzCIO^M ) P G E 2 (lO^M ) PG E2 (1 0 ',0M) Figure 3.6. Tlie effect of PGE2 on LPS induced B7 expression in thioglycollate elicited peritoneal macrophages. 106 cells were cultured in the tissue culture medium alone, in the presence of 10 pg/ml LPS, or LPS together with indicated concentrations of PGE2. The cells were collected after 18 h culture and the B7 expression was measured by FACS. 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.4. Discussion Ligation o f CD40 was first found to costimulate B cell proliferation and to provide an essential signal for the initiation of immunoglobulin isotype switching. The importance o f CD40-CD40L interactions in a functioning humoral immune system was highlighted by die discovery that a defective CD40L gene is responsible for X-linked hyper-IgM syndrome, an immunodeficiency characterized by the absence o f circulating IgG or IgA. and by the absence o f germinal centers (Callard et al.. 1993). Although the initial studies on CD40 focused on its role in humoral immunity, several observations suggested that CD40-CD40L interactions play a broader role in the immune system. Humans (Callard et al.. 1993) and mice (Soong et al.. 1996) with defective CD40L genes display an increased susceptibility to disseminated infections by microorganisms that would usually be controlled by cell-mediated immunity. CD40 expression is not restricted to cells of the B cell lineage, it is also found on dendritic cells, monocytes/macrophages and fibroblasts. Crosslinking o f the CD40 molecules on these cells has been shown to induce functional changes that contribute to cell-mediated inflammatory responses including induction o f cytokine synthesis and up-regulation of the expression o f costimulatory and adhesion molecules (Stout and Sutties. 1996). A possible role of CD40 ligation on the surface o f monocytes has been studied extensively by using membranes from activated CD4 T cells (Wagner et 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. al., 1994), use of crosslinking by antibodies to CD40. and CD40L transfected cells or soluble CD40L, sgp39 (Kiener et al., 1995). The cytokine profile produced by the stimulated monocytes is broad. The different results obtained were a reflection of: (1) the degree of expression of CD40L: (2) the presence or absence o f potential membrane adhesion molecules; or (3) the secretion o f costimulatory cytokines by live cells. All the experiments mentioned above did not measure the B7 expression upon ligation o f CD40 except one in which sgp39 was studied (Kiener et al.. 1995). In that experiment, a small increase in B7-2 was found after 24 h and a significant increase in the level of B7-2 was observed after 96 h. The crosslinking o f CD40 by anti-CD40 antibody should have an effect similar to that o f sgp39. However, in this study no elevated B7 expression on thioglycollate elicited peritoneal macrophages was detected. This may reflect differences between the two cell populations. Peritoneal macrophages recruited by inflammatory signals may have changed their function when they differentiated from the circulating monocytes. To confirm this explanation, in future experiments monocytes should be cultured in an anti-CD40 coated plate, and after various time points, the B7 expression should be measured. The cytokine production by peritoneal macrophages after treatment with immobilized anti-CD40 should also be examined. However, data from B cells clearly demonstrated that the immobilized anti-CD40 was effective in the induction o f B7 expression. 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The interaction between CD40 and CD40L. by activating APCs. plays a crucial role in the activation of T cells. Dendritic cells are implicated in early T cell priming events and the CD40/CD40L interaction induces costimulatory activity on DCs, which is the second signal required for the initiation of a T cell response (Caux et al., 1994). In addition, the CD40/CD40L interaction may result in the production o f IL-12 by DCs (Celia et al., 1996) and macrophages (Shu et al., 1995). IL-12 is a critical cytokine that favors maturation o f Till cells and development o f cell-mediated immunity, as opposed to Th2-mediated humoral immunity. Thus, ligation of CD40 on DCs could contribute to the development and maintenance o f a Thl-type response (Macatonia et al.. 1995). The CD40/CD40L interactions play a significant role in the transition o f resting B cells to become competent professional APC by up-regulating B7 expression on their surface. In contrast, the CD40 mediated activation o f macrophages is likely to be more important for their roles as cytotoxic effectors. The effector functions include the production of cytokines like IL-lcc/'p, IL-6. IL-8. IL-10. IL-12. TNF- a , IFN-y, the generation of nitric oxide, and the production o f metalloproteinase (Stout and Suttles, 1996). Compared with B cells and dendritic cells, whose activation is very dependent on the cell contact with T cells, macrophages are easily activated by signals from microbial pathogens, because they have receptors on their surface to recognize the pattern o f microbial structures on the pathogens. This unique feature of macrophages enables them to be the link between innate and adaptive immunity. 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Not much B7-1 was detected in all experiments presented here. This should be attributed to the low level expression o f B7-1 on these cells, and not to the detection method, because a large amount o f B7-1 was detected in the positive control using B7-1 transfected CHO cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER FOUR FUNCTIONAL COSTIMULATORY ACTIVITY OF MACROPHAGES Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1. Introduction The regulation o f immune responses depends on multiple interactions between APCs and T cells. These interactions are mediated by surface molecules including M HC antigens and costimulatory molecules B7-1 and B7-2. In the preceding chapters, the expression and regulation o f B7-1 and B7-2 on macrophages have been described. In this chapter, the function o f B7 on macrophages as costimulators of T cell proliferation was studied. To ensure that a T cell response is induced, a sufficient number o f TCR and CD28 molecules have to be engaged by their corresponding ligands, peptide- MHC complex and B7. Valitutti and his colleagues (Valitutti et al., 1995) tried to use TCR/CD3 down-regulation to estimate TCR occupancy in vivo, because in the T cells TCR/CD3 complexes are internalized and recycled constitutively. To determine the extent o f TCR down-regulation induced by specific antigens, they constructed a conjugate between T cell clones and autologous Epstein-Barr virus (EBV) transformed B cells pulsed with different concentrations o f the antigenic peptide and measured the level o f TCR/CD3 complexes. Their results showed that a single complex could serially engage and trigger up to — 200 TCRs, and therefore an extremely small number o f peptide-MHC complexes can engage a high number o f TCR molecules and transduce an activation signal. Furthermore, their results showed that integrated TC R occupancy was proportional to the biological response o f the T cells as demonstrated by IFN-y production. 75 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Costimulatory signals are crucial in T cell activation, but how many such molecules are sufficient, together with the TCR signal, to trigger the T cell response has not been elucidated. For the detection and analysis o f the presence o f specific proteins on the cell membrane or intracellularly. ELISA, western blot, northern blot, or even more complicated methods have been applied (Filippini et al., 1998). However, these assays provide, at best, semiquantitative information. Radioimmunoassay is an alternative to provide quantitative information, but non radioactive methods are preferable. During the last two decades, a variety of methods have been developed to achieve quantitation using flow cytometry. The goal o f flow cytometric quantitation of immunofluorescence intensity is to enumerate the number o f certain molecules on (or in) cells o f a given population. These methods are based on the detection of the molecules (antigens) o f interest using monoclonal antibodies (mAb) and the quantification o f mAb binding using a calibration system based on standard beads (Gratama et al.. 1998). Flow cytometry is a fluorescent detection system that can detect a fluorochrome bound to a particle. A particle may be anything from a complex cell type to a simple microbead or microsphere (Fay et al.. 1991; Fulwvler and McHugh, 1990). Using different monoclonal antibody-fluorochrome conjugates, one can detect particles bound with multiple antibodies within a single experiment. If the fluorescent label is bound directly to a receptor on a particle and the binding reaction is stoichiometric, then the fluorescent intensity is directly proportional to the number o f receptors per particle. The number o f receptors per 76 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. particle can be converted to the antibody binding capacity (ABC) o f each particle. The ABC has been easily determined by microbeads. There is a set of commercially available beads contains a mixture o f five populations of microbeads, including four populations that express different calibrated binding capacities of mouse anti-rat IgG monoclonal antibody on their surface and a population of blank beads which has no specific binding capacity for rat IgG to provide an indication o f the detection level o f the instrument. All of the microbeads are o f the same size (8-9 pm diameter) to provide convenient single gating. In order to quantitate the antibody binding capacity of the cells, the Quantum Simply Cellular beads are labeled with the same antibodies used to label the cells. A calibration plot of the binding capacity vs. the respective mean channel number o f the gated microbead peaks is constructed. The binding capacity o f the cell is derived from the calibration plot using the mean channel number o f the cells (Zagursky et al.. 1995). In this chapter, the number o f B7 molecules expressed on macrophages determined by the microbead was correlated with their costimulatory activity for T cell proliferation. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.2. Materials and Methods 4.2.1. T cell preparation T cells were prepared from spleen cells o f BALB/c mice. The spleen was taken from one sacrificed mouse and a suspension o f single cells was prepared. The red blood cells were removed by hypotonic lysis with water and the remaining cells were resuspended in 10 ml TCM and cultured in a 60x15 mm petri dish at 37°C in a humidified incubator for 2 h. The non-adherent cells, which are mainly T cells and B cells, were collected and transferred to a petri dish that had been coated with 100 pg/ml goat anti-mouse Ig. After 30 min at room temperature, the dish was swirled for 30 sec and the non-adherent cells were collected and transferred to a second petri dish that had been precoated with the same amount o f goat anti-mouse Ig. This procedure was repeated two times and more than 95% o f the resulting cells were T cells as detected by positive staining for thy 1.2 by FACS. 4.2.2. Immunofluorescent staining of microbeads The microbeads were purchased from Flow Cytometry' Standards Corporation (FCSC, San Juan, Puerto Rico). The bead suspension was well agitated by vortexing briefly and one drop (about 50 pi, containing ~104 beads) was added to a 5 ml polystyrene round-bottom tube. 1 ml o f staining buffer was 7S Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. added to the tube, centrifuged at 1600 RPM for 1 min. the supernatant was removed by aspiration. 25 pi of 320 pl/ml GL1 F(ab')2 (anti-B7-2. rat IgG2a) was added steadily and rapidly to the beads while vortexing to obtain the tightest distribution. The beads were incubated at room temperature for 1 h. After washing three times with staining buffer. 20 p .1 o f 1:5 FITC goat F(ab')2 anti-rat IgG was added to the beads in the same way as the primary antibody was added and incubated at 4°C for 30 min in dark. The beads were washed three times again and resuspended in 500 pi of staining buffer, ready to be analyzed. 4.2.3. Construction o f standard calibration curve The Quantum Simply Cellular beads contain a mixture o f five populations of microbeads, a blank and four populations that express different calibrated binding capacities for rat IgG monoclonal antibodies. To obtain a standard curve, the microbeads stained as described in 4.2.2 were run in the FAC Scan (Bechman Dickson) flowcytometer at a flow rate between 200-600 events/sec. A live gate on forward and side scatter was set around the singlet population o f the microbeads and 10,000 events were counted. A histogram of the fluorescence was generated. The mean fluorescence intensity channel for each o f the five populations, determined from the histogram, and their corresponding binding capacities were entered into the QuickCal® program, an analysis software that accompanies the microbeads, a standard calibration curve of antibody binding 79 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. capacity (ABC) vs. histogram channel was generated and performance parameters such as square coefficient of the determination (R-Sqrd). average residual percent (AvgRes%), and threshold, etc., were calculated from the standard curve. 4.2.4. Determination of binding capacity o f anti-B7 antibodies to macrophages Resident or thioglycollate elicited peritoneal macrophages were purified by adhesion and cultured in the TCM alone or in the presence of indicated concentrations o f LPS for 18 h. The cells were collected, stained and analyzed by FACS as described in 2.2.5. The values of the mean fluorescence were entered into the QuickCal® program and the corresponding binding capacity was calculated from the standard calibration curve. 4.2.5. Proliferation assay of T cells The potency o f B7 on macrophages to costimulate T cell proliferation was determined by anti-CD3 induced T cell proliferation as measured by the thymidine incorporation of T cells. Purified thioglycollate elicited peritoneal macrophages were cultured with TCM alone or in the presence o f LPS for 18 h at 37°C. The cells were then washed twice with PBS and fixed with 0.5% paraformaldehyde in PBS at room temperature for 30 min. The cells were then washed three times with TCM containing 10% FCS, and kept in the same medium at 37°C until used. 80 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Purified spleen T cells (lCP/well) were cultured with varying numbers o f macrophages in the presence o f purified anti-CD3 antibody YCD3-1 at 37°C in 96-well microtiter plates. 72 h later, 1 pCi o f [JH] thymidine was added to the culture and incubated at 37°C for another 18 h. The cells were harvested and analyzed by scintillation counting. Data were expressed as mean CPM o f [JH]thymidine incorporation by triplicate cultures. In the inhibition assay, the anti-B7-l and anti-B7-2 antibodies were added to the culture at the beginning o f the experiment. 4.3. Results 4.3.1. Quantification o f B7 expression on resident and thioglycollate elicited peritoneal macrophages The B7 expression on resident and thioglycollate elicited peritoneal macrophages was quantitated by using microbeads. Figure 4.1 shows the histogram obtained from the microbeads used to determine the number of B7 on resident peritoneal macrophages and Figure 4.2 shows the standard calibration curve and parameters that indicate how reliable the measurement was. The response of B7 expression to LPS was demonstrated in Figure 4.3. The B7 expression was expressed as the difference o f the mean channel values of fluorescence between sample and control in panel a, while the values were 81 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. converted to the binding capacity o f anti-B7-l or anti-B7-2 antibodies to the macrophages in panel b. There was a certain amount of B7 expressed constitutively on peritoneal macrophages which is the background o f all other measurements. However, the B7-2 expression still responded to LPS in a dose- dependent manner above the background level and the expression reached the saturation level when LPS was used at 10 pig/ml. The histogram and the standard curve used to calculate B7 molecules on thioglycollate elicited peritoneal macrophages are shown in Figure 4.4 and Figure 4.5, respectively. The expression o f B7 responded to LPS in a dose-dependent manner as shown in Figure 4.6. The B7 expression on both resident and elicited peritoneal macrophages responded to LPS similarly except that the elicited macrophages had a higher level o f B7-2 expression. There were about 15.000 B7-2 molecules on each resident peritoneal macrophage activated with 10 (.ig/ml LPS, and approximately 22,000 on thioglycollate elicited macrophages. 4.3.2. Costimulation by macrophages of anti-CD3 induced T cell proliferation Because it is relatively easy to get enough cells from the peritoneal cavity after thioglycollate induction, and because LPS stimulates these cells to express a wide range o f B7 levels, thioglycollate elicited peritoneal macrophages were used in this study. Several factors influenced the proliferation o f a fixed number o f naive T cells from spleen (lO3 cells/well). The effects o f the anti-CD3 antibody concentration, the number o f macrophages, and the concentrations of LPS on B7 82 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. expression are summarized in Figure 4.7. A low concentration o f anti-CD3 antibody (100 ng/ml) could not induce the T cell proliferation. When the antibody concentration reached 500 ng/ml. the proliferation o f T cells was dependent on the number o f macrophages and the concentration o f LPS used to activate the macrophages. The higher the concentrations o f the LPS. the higher the degree T cell proliferation. Similarly. T cell proliferation was closely correlated to the number o f macrophages in the culture when the cell density was low. When the number o f macrophages increased to 3x1 O'/well. T cell proliferation was inhibited. Too many macrophages in the culture may shift the balance to inhibitory signals derived from macrophages, and result in the decreased proliferation o f T cells. The dependency o f T cell proliferation on the concentration o f LPS used to activate macrophages was studied in more detail. In each well. 10' T cells were co-cultured with O.SxlO3 macrophages that had been treated with indicated concentrations o f LPS in the presence o f 500 ng/ml anti-CD3 antibody. Figure 4.8 shows the response o f T cells. Consistent with the B7-2 expression on macrophages in Figure 4.6. T cell proliferation correlated with the LPS concentration in a dose dependent manner. However, the contribution o f other molecules on macrophages whose expression may have also changed in response to LPS could not be excluded from this experiment. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 8 O ■ 3 3 O c o O M l 0 200 € 0 0 SCO F L t •H eight rkcr E vents % Gated % Total Mean P e a k P eak Ch Ail 20000 100.00 100.00 383.41 82 628 M 1 4069 20.34 20.34 619.31 82 628 M2 4348 21.74 21.74 474.18 68 475 M3 6057 30.26 30.28 336.00 72 339 M4 4687 23.43 23.43 207.13 54 255 Figure 4.1. Histogram o f microbeads staining. The beads were stained and analyzed as described in Materials and Methods. This set of beads and resident peritoneal macrophages were run under the same instrument settings. S4 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o ffl < o o § r o - Calibration Plot O “ O O o = Or t 23050 X 130441 < -} ^ _ 40487 .X w . « - m x O o 11878 - « / 37673 O , § _ ' X C 0 _ / 11SS5 - 367-4 y C _ it: y 282 8 m > * — * o o E rf 2 r * A » A f D a ta P o n ts o O' n L C < _ — a«s/ Ft; C B la n k Q S C M ic ro b c a d o o H) 60 120 180 240 Histogram Channel Zero Chan Value Max Chan Value I ---------------------------------------------------1 650 ABC Units 4150455 Perform ance Parameters Acceptable Range Channels: M IN M A X R-Sqr. 09984 0.995 1.000 Blank 30 00 Av Res %: 4.96 0.0 5 0 Bead 1 207 00 Detection Threshold: 840 0 1000 Bead 2 336.00 Coef of Response. 67.01 59.0 69.0 Bead 3 474 00 Log Amp Decades: 3.82 3.7 4 3 Bead 4 619.00 Figure 4.2. Standard calibration curve derived from Figure 4.1. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a 120 c C O C D E a > a c O ) o c n E o L U ■ B7-1 E 3 87-2 LPS (jig/rrt) o x " a ? . o * 0 5 0 2 3 o 0 2 o E C D 20 , 15 - i 10 i 0 -u o > * > x 0 O o o o o J o ■ B7-1 0 B 7 -2 LPS (pg/ml) Figure 4.3. Expression o f B7 on resident peritoneal macrophages. The level of B7 was expressed as the difference mean fluorescence intensity between sample and control in panel a. The fluorescence value was converted to number o f B7 molecules on each cell in panel b. 86 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. © o o o * o M 4 to M 3 © -r M l SOO o 400 0 2 C 0 600 rLI-Heicjht Marker E vents % G ated % T oral Mean P eak Peak Ch All 15000 100.00 100.00 415.99 76 650 M 1 3067 20.45 20.45 643.03 75 650 M2 2916 19.44 19.44 506.30 57 506 M3 4702 31.35 31.35 379.94 53 371 M4 3560 23.73 23.73 255.41 43 271 Figure 4.4. Histogram of microbeads staining. The beads were stained and analyzed as described in Materials and Methods. This set o f beads and thioglycollate elicited peritoneal macrophages were run under the same instrument settings. 87 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. o C D < o C O CL C O 0 0 1 c “ O c C Q > » o £ C < o o o _ o - o - o o Ot- o — Calibration Plot 40487 I 123050 130731 ,v f— F 773 3674 11576 y ^ l l T A O ,rf ' 3801 y 37265 "] Data Points — 3 e s t R t - > Blank GSC Microbead o o '- 60 120 130 Histogram Channel 240 Zero Chan Value Max Chan Valuei l ~ 1 " “ 373 ABC Units - -1 4105406 Performance Parameters Acceptable Ranae Channels: M IN M A X R-Sqr: 0.9982 0.995 1.000 Blank 80.00 A v Res % : 4.70 0.0 5.0 Bead 1 255 00 Detection Threshold: 773 0 1000 Bead 2 379.00 Coef of Response: 63.10 59.0 69.0 Bead 3 506.00 Log Amp Decades: 4.05 3.7 4.3 Bead 4 644 00 Figure 4.5. Standard calibration curve derived from Figure 4.4. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. a 25 , ■ B7-1 0 B7-2 LPS (p.g/nrl) Figure 4.6. Expression of B7 on thioglycollate elicited peritoneal macrophages. The level of B7 was expressed as the difference mean fluorescence intensity between sample and control in panel a. The fluorescence value was converted to number of B7 molecules on each cell in panel b. 89 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25000 - 1 20000 - 15000 - Q . ° 10000 - 5000 - 0.3 3 0.1 1 LPS Anti-CD3 (jig/m l) (ng/m l) 10 10 0.1 o 500 1 0 0 500 500 Number of Macrophages (10 ) Figure 4.7. Dependency o f T cell proliferation on number of macrophages and concentrations of anti-CD3. Various numbers o f LPS treated thioglvcollate elicited peritoneal macrophages and 103 T cells were co-cultured in the presence o f 100 or 500 ng/ml anti-CD3 antibody. 72 h later 1 pCi of [JH]thymidine was added to each well and cultured for another 18 h. Cells were harvested and [JH]thymidine incorporation was measured in a (3 counter. 90 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25000 i 20000 15000 CL o 10000 5000 0.1 0.001 10 0 0.01 1 LPS (^ig/ml) Figure 4.8. T cell proliferation correlates with B7 level on macrophages. Thioglycollate elicited peritoneal macrophages were cultured with LPS at indicated concentrations or 18 h. The macrophages were then washed and fixed with 0.5% paraformaldehyde. 10D treated macrophages and the same number of T cells were co-cultured in the presence o f 500 ng/ml anti-CD3 antibody. T cell proliferation was measured as [JH]thymidine incorporation as described in Figure 4.7. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 30000 25000 - 20000 - c l 15000 - 10000 - 5000 - 0.1 10 0.01 1 0.00001 0.0001 0.001 Anti-B7 Antibody (ng/ml) Figure 4.9. Inhibition of T cell proliferation by anti-B7 antibodies. Thioglycollate elicited peritoneal macrophages were pretreated with 10 ug/ml LPS for 18 h and fixed with 0.5% paraformaldehyde. 5xl04 macrophages were cultured with 103 T cells in the presence o f 500 ng/ml anti-CD3 antibody. [JH]thymidine incorporation was measured as described in Figure 4.7. 92 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 25000 i 20000 - C D o 15000 - 10000 - CQ 5000 - o i 3 0.00001 0.0001 0.001 0.01 10 0.1 1 -B7-1, Blocked ■ B7-2, Blocked -B7-1. Free - B7-2, Free Anti-B7 Antibody (ng/ml) Figure 4.10. Measurement o f the average number o f B7 molecules on each macrophage. Thioglycollate elicited peritoneal macrophages were treated with 10 pg/m l LPS for 18 h. The B7 expression of the macrophages was measured by using the indicated concentrations of anti-B7-l or anti-B7-2 antibodies. The mean fluorescence intensity was converted to the number of B7 molecules occupied by the antibody using the standard calibration curved obtained from Quantum Simply Cellular microbeads. The free B7 molecules available for interacting with CD28 on T cells was calculated as demonstrated in Table 4.1. 93 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4.1. Measurement o f B7 on thioglycollate elicited peritoneal macrophages [Antibody] M FIa Detected number Actual num ber of N um ber of (Hg/ml) of B7/cellb B7/ceIlc free B7/ceIld B7-1 B7-2 B7-1 B7-2 B7-1 B7-2 B7-1 B7-2 10 424 512 17677 39355 318 21996 0 0 1 425 495 17839 33717 480 16358 0 5638 0.1 422 441 17359 20633 0 3274 318 18722 0.01 422 422 17359 17359 0 0 318 21996 0.001 419 420 16891 17046 -486 -313 0 21496 0.0001 423 425 17517 17839 158 480 160 21516 0 422 422 17359 17359 0 0 318 21996 a: mean fluorescence intensity. b: the number o f B7 molecules on each cell directly converted from MFI values and before background value was subtracted, c: the number o f B7 molecules occupied by detecting antibody. The value was calculated by subtracting the background value (17359) from the detected number of B7 molecules per cell, d: the number o f free B7 molecules on each cell. The value was calculated by subtracting the number o f B7 molecules occupied by the detecting antibody from the actual number o f B7 molecules detected by antibody at the saturation concentration, 318 for B7-1 and 21996 for B7-2, respectively. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.3.3. Inhibition o f T cell proliferation by anti-B7 antibodies The involvement o f B7 on macrophages in the anti-CD3 induced T cell proliferation was directly demonstrated in Figure 4.9 by using anti-B7 antibodies to block the interaction between B7 on macrophages and their receptor CD28 on T cells. With increasing concentrations o f the blocking antibodies, the proliferation o f T cells decreased. However, anti-B7-l antibody and anti-B7-2 antibody exhibited different inhibition capacity. Anti-B7-1 antibody only inhibited the proliferation to a moderate degree, even at a concentration as high as 10 pg/ml. When the concentration decreased to 0.01 pg/ml, the anti-B7-l antibody did not have any effect on T cell proliferation. In contrast, at the same concentration, the anti-B7-2 antibody still could inhibit the proliferation almost completely. The lower inhibitory effect o f anti-B7-l antibody may be due to the low level expression o f B7-1 on macrophages. As calculated in Table 4.1, there were only a few hundreds B7-1 molecules on each o f the LPS activated macrophages, compared with more than 20,000 B7-2 molecules on the same cell. How many B7 molecules were blocked by anti-B7 antibodies and how many molecules were still available for interacting with T cells were calculated in Table 4.1 and illustrated in Figure 4.10. Comparing the data in Figure 4.9 and 4.10, when the free B7-2 molecules that can interact with CD28 on T cells were more than 20,000 on each macrophage, T cell proliferation could be triggered. However, a discrepancy exists between the two figures. For example in Figure 95 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.9, when the anti-B7-2 antibody was used at 0.01 (ig/ml, the T cell could not proliferate, although in Figure 4.10, at this concentration o f blocking antibody, the free B7-2 molecules had reached 20,000/cell. The insensitivity o f the FACS analysis may be responsible for the discrepancy. The number o f B7 molecules on each macrophage was calculated from the standard calibration curve of microbeads and the mean fluorescence channel determined by FACS analysis of the cells after staining with various concentrations o f anti-B7-l or anti-B7-2 antibodies. 10 jrg/ml o f anti-B7-2 was the saturation concentration used to measure B7-2 on LPS activated macrophages as determined in a preliminary experiment. When the anti-B7-2 antibody was used at concentrations lower than 0.01 (xg/ml, the FACS analysis could not differentiate the staining quantitatively, although the difference m ay exist (Table 4.1.). This resulted in the large plateau in Figure 4.10 and the discrepancy between Figures 4.9 and 4.10. 4.4. Discussion Optimal T cell activation requires two signals, one initiated through the TCR-MHC-antigen peptide complex and the other through costimulatory molecules, particularly CD28 interacting with B7. It has been shown that CD28 recognizes two different ligands, B7-1 and B7-2, which are related but distinct costimulatory molecules. The role o f B7-1 and B7-2 in costimulating T cell 96 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. costimulatory molecules. The role o f B7-1 and B7-2 in costimulating T cell activation is still controversial. The data presented here demonstrated that LPS enhanced B7-2 expression in thioglycollate elicited peritoneal macrophages which correlated with increased macrophage costimulatory activity for purified T cells to anti-CD3 induced proliferation. Addition o f anti-B7-2 monoclonal antibody to the T cell culture in vitro blocked the enhanced costimulatory ability of macrophages, whereas anti-B7-l antibody had only a minor effect. In addition, the B7-2 level was always higher than B7-1 and no elevation o f B7-1 expression by LPS were observed. It has been demonstrated that cholera toxin (CT) enhances the costimulatory activity of bone marrow-derived macrophages (BMM) in vitro by selectively up-regulating B7-2 expression on these cells, and stimulates a predominantly Th2 response in vivo (Cong et ah. 1997). Consistent with this result, another group showed that bone marrow-derived macrophages selectively and strongly up-regulate expression o f B7-2 after infection with Trypanosoma cruzi (Frosch et ah, 1997). As a consequence o f the increased B7-2 expression, the infected macrophages are able to induce proliferation of splenic CD4 T cells in the presence of anti-CD3 antibody with much higher efficiency than uninfected macrophages. The costimulation of macrophages could be inhibited by an antibody to B7-2. Furthermore, costimulatory activity for established T cell clones o f Thl and Th2 phenotype was also strongly enhanced in T. cruzi infected bone marrow-derived macrophages. 97 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. There is a difference in the dependence on costimulation between Th 1 and Th2 cells. Th2 responses appear to be more dependent on CD28 costimulation during their initiation, but are relatively less so for their maintenance. Conversely, the initiation o f Thl responses appears to be less dependent on CD28 costimulation, but once established, the T hl responses seem to require costimulation for their maintenance. This suggests that B7-2. as the primary costimulatory ligand present in unprimed naive animals, may play a critical role in the initiation o f the Th2 response, whereas B7-1 may be more important in the maintenance o f the T hl response (Bluestone, 1995; Thompson. 1995). In contrast to the results obtained from CD4 T cells or the whole T cell population prepared from lymph nodes discussed above. B7-1 was found to be a stronger costimulator than B7-2 for induction o f proliferation and IL-2 production by naive CD8 T cells from TCR transgenic mice. This difference appeared to be quantitative rather than qualitative. In contrast to those o f nai've cells, the responses of primed T cells to costimulation by B7-1 and B7-2 were quantitatively sim ilar (Fields et al., 1998). The results presented here, according to the discussion above, indicated that after the macrophages are infected with Gram-negative bacteria, whose major component of cell wall is LPS, a Th2 response against the infection will be dominant. The quantitatively different requirement of B7-1 and B7-2 in the activation o f T cells and T cell phenotypes may be one mechanism o f the immune system to control its responses to invading pathogens. 98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The number o f B7 molecules on macrophages was determined using flow cytometry and standard microbeads. The estimation of antigen molecules per cell using this method is rapid and convenient. The data presented here were calculated by using indirect staining. The enumeration by either a direct or an indirect method should be the same if the following conditions are met. First, one antibody molecule should bind to one cell surface antigen. Second, the conformation^ affinity and avidity o f the binding should be the same on anti immunoglobulin coated beads as it is on cells. And lastly, the valency and chemistry o f monoclonal bonding should be the same on cells as they are on beads (Dawson et al.. 1991). To ensure the accuracy o f the determination by this system, both the standard beads and cells should be stained with fluorescence to saturation level. Variability between the calculated number and the actual number may be introduced if only a portion o f the available binding sites on the microbeads are bound with antibody, while all o f the available receptors on the cells are bound (or vice versa). This problem could be avoided if the saturating concentration and an adequate volume of antibodies are used. Vortexing the tubes while adding antibody ensures contact of cells with antibody. Another possibility is that the binding affinity or avidity of the antibody varies depending on whether it is bound to cells or anti-rat IgG coated beads. The fact that the F(ab')2 fragment o f the antibody binds to the cells and the other parts (Fc) of the antibody bind to the beads may be the reason of inducing the difference between actual number and 99 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. measured number o f molecules per cell. Selection o f an appropriate fluorophore is also important for achieving accurate determination. PE labeled antibody is superior over FITC labeling because FITC is associated with self-quenching and interference with cellular auto fluorescence (Dawson et al.. 1991; Gratama et ah. 1998). Strictly, the use o f microbeads determines the Antibody Binding Capacity (ABC). This means that the standards labeled with a specific conjugate antibody have the same fluorescence intensity as that o f the same number o f specific conjugate antibodies bound to cells. This ABC unit is not a direct measurement o f cell receptors, but rather a measurement o f the antibody binding capacity o f the cell for the labeled reagent. The ABC may not reflect the true number o f cell receptors due to, for example, non-specific binding, multivalent antibody interactions, steric hindrance, and hidden or incomplete binding sites. ABC reflects the result of the actual chemical measurement, that is, antigen-antibody interaction. The ABC measurement is only valid when the standards are labeled to saturation with the same antibody (Schwartz et al., 1996). In summary, the results presented in this chapter demonstrate that LPS selectively up-regulates B7-2 expression on peritoneal macrophages, but not B7- 1. The increased B7-2 expression is closely correlated with the costimulatory activity o f these cells for T cell proliferation. Approximately 20,000 B7-2 molecules on each macrophage is sufficient to induce T cell proliferation. The 100 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ratio o f macrophages to T cells, the concentration o f anti-CD3 antibody (or the strength o f TCR signal) also contribute to optimal T cell proliferation. 1 0 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER FIVE DEVELOPMENT OF A CELL ELISA METHOD TO MEASURE B7 EXPRESSION Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.1. Introduction B7 molecules on antigen presenting cells (APCs) provide costimulatory signals that are important to activate T cells and thus initiate an immune response. In the present study and in almost all literature reports, the expression of B7 molecules on APCs has been measured by flow cytometry. Flow cytometry, first introduced in the early seventies (Loken and Herzenber, 1975). employs instrumentation that scans single cells flowing past excitation sources in a liquid medium. The technology is unique in its ability to provide rapid, quantitative, and multiparameter analyses on single living cells. Measurement o f visible and fluorescent light emission allows quantitation of antigenic, biochemical, and biophysical characteristics of individual cells. The first step of performing flow cytometry experiments is to stain cells with fluorescent reagents, either directly or indirectly. In direct immunofluorescence staining, cells are treated with an antibody that has been conjugated to a fluorochrome. In indirect staining, the primary reagent is not labeled but is detected by a second fluorochrome-labeled reagent. This second reagent may be an antibody with specificity for the first antibody. Alternatively, the avidin-biotin system can be used, whereby an antibody is conjugated to biotin and is detected with fluorochrome-labeled avidin. This system was used in detecting CD40 expression on macrophages in section 3.2.3. Indirect immunofluorescence offers several advantages over direct immunofluorescence. 103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For instance, biotinylated antibodies can be detected with avidin conjugated to any number o f different fluorochromes that are inexpensive, of high quality, and widely available from a number o f commercial sources. In immunofluorescence using two antibodies, detection o f a first antibody that is unpurified and unlabeled can be achieved by using a second purified, labeled antibody. One secondary antibody can be used for the detection of many primary antibodies with different antigen specificity. In addition, indirect immunofluorescence often amplifies the fluorescence signal. The goal in immunofluorescence staining is to attain a high specific signal with minimal background. The background fluorescence of cell auto fluorescence results from light emitted by naturally occurring intracellular materials excited at the wavelength used to excite chemically linked fluorescent probes. The most troublesome sources of autofluorescence are in hematopoietic cells that are excited at 488 nm. Because the peak wavelength o f autofluorescence emission is near 560 nm (yellow), it significantly interferes with measurements o f fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE). Autofluorescence is significantly higher for in vitro cultured cells, cells with high granule contents, like macrophages and tumor cells. Auto fluorescence can be reduced by using cells in an exponential growth phase, appropriate optical filters, and reagents that have been conjugated with red fluorochromes. Another source of background is caused by the binding o f IgG antibodies to Fc receptors regardless o f their antigen specificity. This problem can be 104 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. prevented if antibodies are prepared as Fab or F(ab?)i fragments, which removes the Fc portion o f the IgG molecule (Parham. 1983). This strategy is more important in die measurement o f B7 on macrophages in this study, because both anti-B7-l and anti-B7-2 antibodies are of the IgG?a isotype, and macrophages express high levels o f Fc receptors. Another approach to reduce nonspecific binding to Fc receptors is to block the binding sites with unlabeled antibodies specific for Fc receptors or with an excess of serum. In this approach, care must be taken to ensure that labeled second antibodies do not recognize the blocking antibodies. Direct staining o f cells with immunofluorescence together with utilization o f anti-Fc receptor antibodies has been applied by some investigators to measure B7 expression on APCs (Hathcock et al., 1994; Stack et al.. 1994). An alternative technique for detection o f cell surface antigen that has sensitivity comparable with that of flow cytometry is enzyme-linked immunosorbent assay (ELISA) (Bartlett and Noelle. 1987). Since their first description in 1971 (Engvall and Perlman. 1971). ELISAs have become the system o f choice when assaying soluble antigens and antibodies. At the same time, replacement of radio-labeled reagents with enzyme-labeled reagents for the quantitative assessment of molecules on the cell surface was first reported by Avrameas and Guilbert (Avrameas and Guilbert. 1971). Factors that have contributed to the success o f ELISAs include their sensitivity, the long shelf-life of the reagents, the lack o f radiation hazards, the ease o f preparation o f the reagents, the speed and reproducibility of the assays, and the variety of ELISA 105 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. formats that can be generated with a few well-chosen reagents. Additionally, no sophisticated equipment is necessary for many ELISA applications, including the screening of hybridoma supernatants for specific antibodies and the screening of biological fluids for antigen content. Cellular ELISA is especially useful for screening cells for expression of antigen, measuring expression o f cell-surface antigens, or screening for antibodies against cellular antigens. In cell-ELISA, the cells are first incubated with antibody specific for the cell surface antigen o f interest. After washing out unbound antibody, the cells are incubated with secondary antibody covalently coupled to an enzyme. Unbound conjugates are washed out and a chromogenic or fluorogenic substrate is added. As the substrate is hydrolyzed by the bound enzyme conjugate, a colored or fluorescent product is generated. Finally, the product is detected visually or spectrophometrically, most commonly with a microtiter plate reader. The amount o f product generated is proportional to the amount o f analysate in the test mixture. A number o f different enzymes have been successfully used in ELISAs. among which alkaline phosphatase (AP), horseradish peroxidase (HRJP) and (3 - galactosidase are most widely applied (Sedgwick and Czerkinsky. 1992). However, when the enzymes HRP and AP are used in cell-ELISA, a high background color formation associated with the endogenous cellular enzyme activity is usually introduced. The spontaneous hydrolysis of o- phenylenediamine, and p-nitrophenyl phosphate, the substrates o f HRP and AP, 106 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. respectively, by spleen cells has been described. In addition, background binding o f secondary antibody conjugated to HRP and AP was observed (Liu et al.. 2000). Attempts were made to inhibit this activity but with limited success. The utilization of p-galactosidase in cell-ELISA was first introduced in 1981 by Cobbold and Waldmann (Cobbold and Waldmann. 1981). This enzyme is not usually found in eukaryotic cells and therefore, is preferable to HRP and AP because the background is very low. Taking the advantage of low background produced by P-galactosidase and the lack of interaction between F(ab')i and Fc receptors that causes non-specific binding of the detecting antibodies to Fc receptor bearing cells, an ELISA method was developed by coupling F(ab’)2 fragments of anti-B7-2 antibody. GL1. to P- galactosidase using a water soluble heterobifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]-cyclohexane-1 -carboxylate (sulfo- SMCC) that has NHS ester and maleimide groups reactive toward amino and sulfhydryl groups, respectively. The purpose o f developing a cell-ELISA method to measure B7 molecules is to provide an alternative approach to confirm the results obtained by flow cytometry. In this study. B7-2 expression on B cells was measured by one step cell-ELISA and the resuit was compared with that by flow cytometry. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2. Materials and Methods 5.2.1. Reagents and antibodies GainmaBind Plus Sepharose and CNBr activated Sepharose 4B were the products o f Pharmacia (Uppsala. Sweden). Immobilized pepsin, bovine serum albumin (BSA, grade V), NaNs, LPS, HEPES. Trizm a base (Tris). HCI. Tween 20. p-mercaptoethanol. Sephadex G-25 and G-200 were purchased from Sigma Chemicals (St. Louis, MO). P-Galactosidase (from E.coli. EIA grade) and chlorophenolred-P-D-galactopyranoside (CPRG, monosodium salt, substrate of and P-galactosidase) were from Boehringer Mannheim (Indianapolis. IN). Sulfosuccinimidyl 4-[/V-maleimidomethyl]-cyclohexane-l-carboxylate (sulfo- SMCC) was from Pierce (Rockford. IL). Rabbit complement was purchased from ICN Biomedicals (Costa Mesa, CA). Goat anti-rat IgG was obtained from Accurate (Westbury, NY), and FITC goat F(ab') 2 anti-rat IgG from Caltag Laboratories (Burlingame, CA). Hybridomas GL1 (rat IgG^a anti-mouse B7-2). RG 7/1.30 (mouse anti-rat IgGia Fc), and 30-H12 (rat anti-mouse Thy 1.2). were obtained from American Type Culture Collection (ATCC. Rockville. MD). 5.2.2. Purification of antibodies Rat anti-mouse B7-2 antibody was purified from GL1 hybridoma culture supernatants. The hybridoma was grown in TCM containing 5% FBS in a 75 cm2 108 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. flask and supernatant was collected every 2 to 3 days. About 2 L supernatant was filtered with a bottle top filter and the pH was adjusted to 7.0 with 1 M HEPES free acid solution. The supernatant was then passed through a GammaBind Plus Sepharose column that had been equilibrated with binding buffer (10 mM sodium phosphate, 0.15 M NaCl. 10 mM EDTA. pH 7.0) and the antibody bound to the sepharose beads was eluted with 0.5 M acetic acid (pH 3.0). The antibody solution was neutralized immediately with 2 M Tris-HCI (pH 11) to pH 7.0. Fractions containing a large quantity of antibody as determined by the absorbance at 280 nm were pooled and dialyzed against 2 L PBS overnight in a Slide-A-Lyzer cassette (Pierce. Rockford. IL). The concentration o f purified antibody was determined using BioRad protein assay kit (BioRad. Hercules. CA) using bovine IgG as a standard. 5.2.3. Preparation o f F(ab’)2 fragments Purified antibody was dialyzed into digestion buffer (100 mM acetate buffer. pH 4.0) and concentrated to 3mg/ml using a Centricon-10 concentrator (Centricon-10, Amicon Inc., Beverly. MA). The immobilized pepsin was reconstituted according to the manufacturer's instruction and added to 2 ml antibody solution at the ratio of 1:20 (w/w). After 7 h of incubation at 37°C. the immobilized pepsin was removed and the antibody solution was dialyzed overnight against PBS at pH 7.0. The antibody F(ab’)i fragment was further purified by affinity chromatography using anti-Fc mAb RG 7/1.30 that had been 109 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. conjugated to a CNBr activated sepharose 4B column following the manufacturer’s instructions. The purity of the F(ab’)2 fragments was assessed by SDS-PAGE. 5.2.4. Synthesis o f the antibody-P-galactosidase conjugate Purified antibody F(ab’)i fragments were transferred into 100 mM phosphate buffer (pH 7) by dialysis and concentrated from 0.86 mg/ml to 5 mg/ml by centrifugation in Centricon-10 tubes. Sulfo-SMCC (1 mg o f dry powder) was added to 0.5 ml antibody solution and incubated at 30°C for 30 min. Excess sulfo- SMCC was removed by Sephadex G-25 chromatography (Econo-Column. 1x30 cm, BioRad) using 100 mM phosphate buffer containing 150 mM NaCl (pH 7.0) for elution. The antibody eluted in void volume was concentrated to 2 mg/ml using Centricon-10 tubes. This maleimide activated antibody (0.5 ml) was mixed with 0.6 mg P-galactosidase dissolved in 0.5 ml of 100 mM phosphate buffer containing 150 mM NaCl (pH 7.0), and incubated at 30°C for 40 min to form the antibody-enzyme conjugate. The reaction was terminated by adding P- mercaptoethanol at a final concentration of 10 pM. The final product was purified by Sephadex G-200 chromatography. Fractions o f 1ml eluate were collected and the absorbance at 280 nm was read in a spectrophotometer (Spectronic 1001, Milton Roy, Rochester, NJ). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.2.5. Assay o f (3-gaIactosidase-antibody conjugate formation by capture ELISA ELISA plates (Immunlon-4. Dynatech Laboratories, Boston, MA) were coated overnight at room temperature with 50 pi of 2.5 pg/ml goat anti-rat IgG in PBS containing 0.05% NaN3. The plates were washed 3 times with PBS containing 0.05% Tween 20 (PBS/Tween 20) and treated with blocking buffer containing 15 mM N aoB^v, 120 mM NaCL, 0.05% Tween 20. 1 mM EDTA. 0.25 % BSA and 0.05 % NaN3, pH 8.5 for 30 min at room temperature and washed with PBS/Tween 20 three times. For the assay. 50 pi o f serial dilutions of antibody-(3-galactosidase conjugate in staining buffer (PBS containing 2% FBS and 0.1% NaN3) were added. The plates were incubated for 2 h at room temperature. After washing three times with PBS/Tween 20. 100 pi o f substrate solution (PBS containing 0.1% NaN3, 1% BSA. 2 mM MgCL. 2 mg/ml CPRG) was added to the plates and the absorbance at 570 nm was read every 3 min. The enzyme activity was calculated as the slop o f the color development over time. 5.2.6. Direct (one-step) ELISA to measure B7 on B cells B cells were isolated from mouse spleen by depleting T cells with anti- Thyl.2 antibody and complement. B blasts were generated by culturing 2.x 106- 4 x l0 6 B cells/ml in the presence o f 10 pg/ml LPS for 18 hours. B ceils or B cell blasts were seeded in wells of a round-bottom 96 well plate at a density of 200,000 cells/well and incubated with 100 pi antibody-enzyme conjugate diluted 111 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. in staining buffer for 30 min on ice. After washing three times with staining buffer, P-galactosidase was assayed as described in 5.2.5. 5.3. Results 5.3.1. Formation of antibody-|3-galactosidase conjugates using a heterobifunctional crosslinker For the formation of antibody-enzyme conjugate using the heterobifunctional crosslinker sulfo-SMCC, a two-step process was employed. The ester group in sulfo-SMCC was first allowed to react with free amino groups in the antibody to form a maleimide-conjugated antibody. At the second step the maleimide group was reacted with free sulfohydryl groups in p-galactosidase to form the final product as shown in Scheme 5.1. Each p-galactosidase molecule has about 12 to 16 free — SFI groups and are ready to react with maleimide groups o f sulfo-SMCC. The enzyme-antibody conjugate was purified by Sephadex G- 200 chromatography and the conjugate was eluted in void volume. For a functional test of conjugate formation, the conjugate preparation was added to ELISA plates coated with goat anti-rat antibody and after incubation and wash, p-galactosidase activity was measured. Since the conjugation partner, anti- B7-2 mAb GL1 F(ab?)2, is a rat antibody, plate-bound enzyme activity can be detected only if conjugation has been successful. The results in Figure 5.1 show 112 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. that when the same amount o f P-galactosidase in either antibody-conjugated or free form was added to the antibody precoated plate, only enzyme in conjugated form displayed activity that increased with the amount o f conjugate added to the assay. Only at high concentrations a low level o f activity was detected with the free enzyme, probably due to some non-specific binding to the plate. The data clearly demonstrated that a conjugate between GL1 F(ab')2 and p-galactosidase had been formed. 5.3.2. Measurement o f B7-2 expression on B cells using anti-B7-2 mAb F(ab?)i- P-galactosidase conjugate and comparison with that using flow cytometry B7-2 expression on ex vivo B cells, cultured B cells, and LPS stimulated B cell blasts was measured by the anti-B7-2 mAb GL1 F(ab')2-P-gaIactosidase conjugate. The data show that freshly prepared B cells express a low level o f B7- 2. After a period in culture the B7-2 level was increased and LPS could further up-regulate B7-2 expression. The measured amount o f B7-2 was proportional to the concentration of the conjugate added to the cells as demonstrated in Figure 5.2. The cell-ELISA data were consistent with data from flow cytometry (Figure 5.3) and with findings by Hathcock et al. (1994) who demonstrated that B cells upregulate B7-2 expression during culture and that LPS is a potent stimulator o f B7-2 expression by B cells. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. N a 0 3 S n h — ;a!actosidase. s n antibod’ n h — C- — 0— 0 antibod’ antibod' O Schem e 5.1. Structure o f sulfo-SMCC and the reactions to form antibody-enzyme conjugate. The sulfo-NHS ester group in sulfo-SMCC first reacts with free amino groups o f the antibody to form reactive maleimide-tagged antibody. At the second step the maleimide group reacts with free sulfhydryl groups in P- galactosidase to form the final product. 114 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.7 i >» -4 -3 -2 -1 0 Log of (3-Gal activity (U/ml) Figure 5.1. Capture ELISA, performed to assay formation o f mAb G Ll F(ab')2- P-galactosidase conjugate. ELISA plates were coated with goat anti-rat antibody and antibody-p-galactosidase conjugate was added at the indicated concentrations (closed symbols). To controls, free p-galactosidase was added (open symbols). After incubation and wash, plate bound enzyme activity was determined. It is expressed as change in absorbance at 570 nm during 6 min. Data are the mean of triplicate determinations. P-Gal: P-galactosidase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 0.8 i ° - 7 i 0.6 j 0.5 - j & 0.4 - | ■ M 0.3 - j o c o o co < <T C D O I C O . 0.2 0.1 0 i 0.2 I ■ in 0.1 0.05 0.025 E 3 blast □ cultured ■ ex vivo p-Gal activity of p-Gal-GL1 -F(ab')2 conjugate (u/ml) Figure 5.2. Measurement of B7-2 expression on ex vivo (freshly isolated) B cells. B cells cultured in medium and B blasts by cell-ELISA using a direct method. Cells (200.000/well) were incubated with indicated concentrations o f GL1 F(ab’)2-p-galactosidase conjugate on ice for 30 min. followed by wash and measurement o f enzyme activity. The B7-2 level on cells is expressed as the change in absorbance at 570 nm during 30 minutes. Data are the mean of duplicate determinations. P-Gal: P-galactosidase. 116 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CO CD 140 120 E 100 0 3 80 0 3 60 C J C /3 0 3 40 ex v iv o cultured blasts ex v ivo cultured blasts Figure 5.3. Comparison of cell-ELISA (a) and flow cytometry (b) to measure B7- 2 expression on ex vivo (freshly isolated) B cells. B cells cultured in medium and B blasts. For cell-ELISA, a direct method was used. Cells (200.000/well) were incubated with 4 p.g/ml o f GL1 F(ab?)2-P-gaIactosidase conjugate on ice for 30 min, followed by wash and measurement of enzyme activity. The B7-2 level on cells is expressed as the change in absorbance at 570 nm during 30 minutes. Data are the mean of duplicate determinations. For flow cytometry, unlabeled GL1 F(ab’)2 fragment was used as primary antibody and FITC-labeled Ffab'L fragment of goat anti-rat IgG was used as secondary antibody. Binding o f secondary antibody in the absence o f primary antibody was subtracted from the data. Data are representative o f four sim ilar experiments. Errors were less than 10% o f values. P-Gal: P-galactosidase. 117 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. c O ° - 8 0 C O < 0.70 J f 0.60 ro 0.50 0 3 9 0.40 C 2 . " O c 0.30 o -9 0.20 G L1 F(ab’ )2 Irrelevant antibody ^ 0.001 0.01 0.1 1 10 100 1000 Concentration of pure antibody (j.ig/ml) Figure 5.4. Competition assay to test the specificity of GL1 F(ab')2-(3- galactosidase. B cell blasts (200,000/well) were incubated with indicated concentrations o f pure GL1 F(ab’)2 antibody or an irrelevant antibody on ice for 30 min. The GLl F(ab!)2-P-galactosidase conjugate was then added to the cells. The enzyme activity is expressed as the change in absorbance at 570 nm during 30 minutes. Data are the mean o f duplicate determinations. p-Gal: p- galactosidase. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5.3.3. Specificity o f the anti-B7-2 mAb F(ab')2-P-galactosidase conjugate In Figure 5.2. the higher B7-2 was detected with the higher concentration o f conjugate was used. To distinguish whether the increased enzyme activity was caused by an increase of B7-2 expression or the non-specific binding of the conjugate to the cell surface, purified anti-B7-2 mAb GL1 and an irrelevant antibody were used to compete out the binding o f the GLl Ffab’E-p- galactosidase conjugate to B blasts. As shown in Figure 5.4. when the concentration o f purified G Ll Ffab7 )? was higher than 1 ug/ml. it could effectively inhibit the binding o f the conjugate to cells. In contrast, irrelevant antibody did not have any effect even at concentration as high as 600 j-ig/ml. As complementary evidence to results in Figure 5.1, this result provides further evidence that the antibody-enzyme conjugate, which is specific for B7-2. is successfully formed by utilizing sulfo-SMCC. 5.4. Discussion An antibody-enzyme conjugate was generated by using a water soluble heterobifunctional linker sulfo-SMCC and has been used to measure B7-2 expression on B cells using cell ELISA. The functional characterization by capture ELISA showed a 1000-fold increased enzyme activity in the conjugate (Figure 5.1). The further functional assay o f this conjugate was done on intact 119 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cells. As shown in Figure 5.2. different B7-2 levels were measured by the conjugate in a dose dependent manner. In order to rule out the possibility o f non specific binding o f the enzyme to cells causing the phenomena seen in Figure 5.2. a competition assay was conducted. Results in Figure 5.4 show the specificity of the conjugate to B7-2 on the cell surface. The comparison o f cell-ELISA and flow cytometry to measure B7-2 expression on B cells was performed and the two methods yielded comparable results. However, the instrumentation and analysis required for ELISA are much cheaper and easier than those of flow cytometry. The advantage o f ELISA to measure cell surface antigen may be even more obvious for adherent cells since the removal o f such cells by trypsinization for flow cytometry analysis could damage some o f the proteins o f interest. The ELISA method described here could be further improved. If the p- galactosidase is coupled to a secondary antibody used in indirect two-step ELISA, the reagent will have a broader application, as long as the primary antibody is from the same species and has the same isotype. The composition and the stoichemistry o f the conjugate need to be further identified. The purification by Sephadex-200 chromatography is still crude and the antibody-enzyme preparation may contain impurities. The measurement of B7 on B cells gave similar results by ELISA and flow cytometry. Indeed ELISA and flow cytometry are two complementary methods. Under certain conditions one is preferable over the other. For example, for initial screening of monoclonal antibodies, flow cytometry is undoubtedly 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. more time consuming than cell-ELISA. Thus, in situations where a monoclonal antibody is sought against a cell population that is available in a purified form. cell-ELISA is definitely quicker and may be at least as sensitive as flow cytometry (Aida et al., 1987). However, in those situations where monoclonal antibodies are sought against relatively minor subpopulations of cells present within a heterogenous mixture, flow cytometry is unmatched for its discrimination power and sensitivity. Additionally, the coordinate expression o f more than one antigen or surface molecule on a single cell can be analyzed simultaneously in one sample preparation using multicolor staining by flow cytometry, which is not easily achieved by ELISA. Many crosslinkers have been used to make antibody-enzyme conjugates for use in enzyme immunoassays (O'Sullivan and Marks, 1981). Glutaraldehyde is one o f the most widely used homobifunctional aldehyde that reacts with amino residues in proteins. Horseradish peroxidase (HRP) and alkaline phosphatase labeled antibodies have been successfully formed by this technique. However, the efficiency is low, heterogeneous and highly polymerized conjugates are found. A(A’ -o-phenylenedimaleimide is a homobifunctioal maleimide that reacts rapidly and selectively under mild conditions with molecules containing sulfhydryl groups. This method has been used efficiently to couple P-galactosidase to antibody with little or no loss of enzyme or antibody activity. The requirement o f free sulfhydryl groups on both enzymes and antibodies limits its application. An improved method is to apply heterobifunctional linkers, such as m- 121 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. maleimidobenzoyl-iV-hydroxysuccinimide ester (MBS). This reagent contains a hydroxysuccnimide ester reacting with the amino groups and a maleimide residue reacting with sulfhydryl residues, respectively, allowing control over the two coupling steps involved in the preparation o f antibody-enzyme conjugate. The reactions are rapid and selective. Compared with the methods mentioned above, the MBS procedure is straightforward and efficient with little or no loss o f enzyme activity' and a relatively small loss o f antibody activity. The problem with MBS is its instability at neutral pH. Due to its water insolubility MBS has to be dissolved in organic solvent before it can be added to protein solution. Succinimidyl 4-[iV-maleimidomethyl]-cyclohexane-1 -carboxylate (SMCC), another heterobifunction linker, has been used to couple antibody to toxins (Morgan et al., 1990), proteins to cells (Christiaansen et al.. 1984). and glucose oxidase to rabbit Fab' (Yoshitake et al., 1979). Sulfo-SMCC as shown in Scheme 5.1, is a water soluble, non-cleavable and membrane impermeable heterobifunctional linker. The cyclohexane bridge gives extra stability to the maleimide reactive group. Y-(4-carboxycyclohexylmethyl) maleimice groups are stable for 64 hours in 0.1 M sodium phosphate buffer. pH 7.0 at 4°C. The addition of the suifo group increases the water solubility and therefore the coupling reactions will occur in water buffers. In contrast, the water insoluble linker MBS may result in precipitation at the first step of the coupling reaction that occurs in organic solvent, such as dioxane. The efficiency, simplicity, 122 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. stability and water solubility o f sulfo-SMCC make it the first choice for forming conjugate between P-galactosidase and GLl F(ab’)2 fragments in this study. The conjugate was prepared under optimized condition collected from the literature describing the synthesis o f other protein-protein conjugates, i.e. high concentration o f antibody, large excess of sulfo-SMCC. neutral pH, controlled temperature and incubation time (Christiaansen et al.. 1984; Idziorek and Klatzmann, 1991; Melton et al., 1993; Yoshitake et al., 1979). The use of P- galactosidase was based on the following considerations: (1) this enzyme is not present in the cell, so the background level is low; (2) the use o f chromogenic substrate m ay increase the sensitivity of the detection; (3) p-galactosidase has free sulfhydryl groups ready to react with the maleimide residues on the crosslinker, and no modification is needed. In summary, a cell-ELISA method was developed to measure B7-2 expression on Fc receptor bearing cells, like B cells. The sensitivity is comparable to that with flow cytometry. The GLl Ffab’j^-P-galactosidase was formed by using a water soluble heterobifunctional crosslinker sulfo-SMCC. This coupling is rapid, simple and efficient. The same procedure could be modified and applied to couple various proteins, including antibodies, enzymes, peptides, etc., generating diverge reagents could be used in basic and clinical research. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER SIX SUMMARY AND PERSPECTIVES Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Costimulatory molecules provide a critical signal for T cell activation and initiation o f an immune response. B7 is one o f the most important costimulatory molecules expressed on professional antigen presenting cells. The results of its expression, regulation, and function on macrophages are reported in this dissertation. The major findings are summarized as follows: 1) B7 is not equally expressed on macrophage subpopulations. Very low expression o f B7 is detected on alveolar macrophages, and a significant amount o f B7-2 is found on both resident and thioglycollate elicited peritoneal macrophages. 2) B7 expression on macrophage subpopulations responds to LPS stimulation differentially. B7-2 expression is in the order o f thioglycollate elicited peritoneal macrophages > resident peritoneal macrophages > alveolar macrophages. 3) LPS induced TN F-a production differs from B7 expression. It is in the order o f alveolar macrophages > thioglycollate elicited peritoneal macrophages > resident peritoneal macrophages. 4) In contrast to dendritic cells and B cells, crosslinking of CD40 on macrophages by immobilized anti-CD40 antibody dose not result in the up- 125 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. regulation o f B7 on this cell population, which implies that the activation o f macrophages is less dependent on signals from T cells, but is more responsive to microbial stimulation. 5) IFN-y, a cytokine released from Thl cells, has similar effect as LPS on up- regulating B7-2 expression on macrophages. GM-CSF. a factor important for the maturation o f macrophages, does not influence the B7 expression on matured peritoneal macrophages. PGEi, an anti-inflammatory factor produced by macrophages themselves, does not abolish the effect of LPS on B7-2 expression. 6) The expression o f B7-1 is low on both alveolar and peritoneal macrophages and does not respond to any o f the stimuli tested. 7) The number o f B7 molecules on each macrophage is estimated by using microbeads and responds to LPS in a dose-dependent manner. 8) The amount o f B7-2 molecules on macrophages is essential for nai've T cell proliferation induced by anti-CD3 antibody. Approximately 20.000 B7-2 molecules/cell is sufficient for optimal T cell proliferation. The number o f macrophages and the macrophage/T cell ratio are also important. 126 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 9) A one-step ELISA method has been developed to measure B7 on antigen presenting cells by coupling F(ab') 2 fragments of anti-B7 antibody to (5- galactosidase with a water soluble heterobifunctional linker sulfo-SMCC. The formation of an antibody-en2yme conjugate by this method is simple, rapid and efficient. Measurement of B7-2 on B cells using ELISA yielded similar result to flow cytometry. Based on the results presented in this dissertation, further research may be focused on the following: 1) What is responsible for the differentiated responses o f macrophage subpopulations to LPS? Is it the different CD 14 expression on these macrophage subpopulations? How is CD 14 expression regulated? 2) To compare macrophage subpopulations in their ability to costimulate T cell activation. What causes the differences, if there are any? The different levels of B7 or some other factors? 3) The regulation o f B7-1 expression on macrophages. 4) The effect of CD40-CD40L interaction on different macrophage functions. 127 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 5) To compare the expression, regulation and function o f macrophages from NOD mice, the animal model of type I diabetes, with macrophage from their disease resistant control. Because macrophages have been shown to be involved the disease development, the comparison may reveal how macrophages contribute to this process and how it can be controlled to prevent and treat disease. 6) To couple (3-galactosidase to F(ab') 2 fragments o f a secondary antibody so that a two-step ELISA that has broader applications can be used to measure B7 on antigen presenting cells. 7) To define the composition o f the antibody-p-galactosidase conjugate. 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Creator Liu, Zheng (author)

Core Title Expression and regulation of B7 on antigen presenting cells

School Graduate School

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag health sciences, immunology,OAI-PMH Harvest

Language English

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Advisor Von Grafenstein, Hermann (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-64468

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